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The Molecular Architecture of Hemidesmosomes as Revealed by Super-Resolution Microscopy
Hemidesmosomes have been extensively studied by immunofluorescence microscopy, but due to its limited resolution, their precise organization remained poorly understood. We studied hemidesmosome organization in cultured keratinocytes by 2- and 3-color super-resolution microscopy. We observed that in the cell periphery, nascent hemidesmosomes are associated with individual keratin filaments and that β4 is distributed along rather than under keratin filaments. By applying innovative methods to quantify molecular distances, we demonstrate that the hemidesmosomal plaque protein plectin interacts simultaneously and asymmetrically with β4 and keratin.Read article -
Practical Guide for Excellent GSDIM Super-Resolution Images
Do you know that most protists and bacteria lack in one feature that each of our body cell has? Our cells are touch and communicate with one another. They send and receive a variety of signals that coordinate their behavior to act together as a functional multicellular organism. Exploring the way of cellular communication and the ways how the cell surface interacts to organize tissues and body structures is of great interest. Kees Jalink and his team of scientists at the Netherlands Cancer Institute (NKI) in Amsterdam obtained new scientific insights into the molecular architecture of hemidesmosomes, cytoskeletal components, cell surface receptors and vesicular proteins with the help of Ground-State-Depletion (GSD)/ dSTORM microscopy. In this interview, Kees Jalink comments on their developments in imaging chambers, buffer conditions and image analysis to get the perfect super resolution image.Read article -
Initiation of Lamellipodia and Ruffles Involves Cooperation Between mDia1 and the Arp2/3 Complex
Protrusion of lamellipodia and ruffles requires polymerization of branched actin filaments by the Arp2/3 complex. Although regulation of Arp2/3 complex activity has been extensively investigated, the mechanism of initiation of lamellipodia and ruffles remains poorly understood. Here, we show that mDia1 acts in concert with the Arp2/3 complex to promote initiation of lamellipodia and ruffles.Read article -
Visualizing Tropoelastin in a Long-Term Human Elastic Fibre Cell Culture Model
Elastin is an essential protein found in a variety of tissues where resilience and flexibility are needed, such as the skin and the heart. When aiming to engineer suitable implants, elastic fibres are needed to allow adequate tissue renewal. However, the visualization of human elastogenesis remains in the dark. To date, the visualization of human tropoelastin (TE) production in a human cell context and its fibre assembly under live cell conditions has not been achieved. Here, we present a long-term cell culture model of human dermal fibroblasts expressing fluorescence-labelled human TE. We employed a lentiviral system to stably overexpress Citrine-labelled TE to build a fluorescent fibre network. Using immunofluorescence, we confirmed the functionality of the Citrine-tagged TE. Furthermore, we visualized the fibre assembly over the course of several days using confocal microscopy. Applying super resolution microscopy, we were able to investigate the inner structure of the elastin–fibrillin-1 fibre network.Read article -
Botulinum Neurotoxin Type-A Enters a Non-Recycling Pool of Synaptic Vesicles
Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity.Read article -
Abstracts of the 6th European Super-Resolution User-Club Meeting
The 6th European Super-Resolution User Club Meeting was held in collaboration with Dr. Timo Zimmermann, CRG, and Dr. Pablo Loza-Alvarez, ICFO, Barcelona. According to the founding principle of the club of keeping close to science, both imaging facilities at the CRG and the ICFO opened their doors to the User Club members, allowing them to explore exciting super-resolution and and nanoscopy applications. The meeting agenda covered highly relevant talks around this year’s central theme “Core Facilities and Super-Resolution Microscopy”, as well as plenty of opportunities to network amongst super-resolution users from different European countries. Here we present the abstracts of the talks held during the meeting.Read article -
Quantifying the Resolution of a Leica SR GSD 3D Localization Microscopy System with 2D and 3D Nanorulers
DNA origami based nanorulers produced by GATTAquant are common standards to test the achievable spatial resolution of super-resolution microscopes. Recently the nanorulers were used to test the performance of the Leica SR GSD 3D microscope.Read article -
"The Leica Digital Light Sheet Module – a Clever Example of Thinking Out of the Box"
Bram van den Broek is a postdoctoral fellow at the Netherlands cancer institute in Amsterdam where he supports the advanced microscopy techniques in the laboratory of Kees Jalink. Working with Leica Microsystems as a collaboration partner for beta-testing of microscopes he enjoys very much.Read article -
Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy
Co-localization analysis is a widely used tool to seek evidence for functional interactions between molecules in different color channels in microscopic images. Here we extend the basic co-localization analysis by including the orientations of the structures on which the molecules reside. We refer to the combination of co-localization of molecules and orientational alignment of the structures on which they reside as co-orientation. Because the orientation varies with the length scale at which it is evaluated, we consider this scale as a separate informative dimension in the analysis. Additionally we introduce a data driven method for testing the statistical significance of the co-orientation and provide a method for visualizing the local co-orientation strength in images. We demonstrate our methods on simulated localization microscopy data of filamentous structures, as well as experimental images of similar structures acquired with localization microscopy in different color channels.Read article -
Abstracts of the 5th European Super-Resolution User-Club Meeting
The 5th Super-Resolution User Club Meeting was held in collaboration with Professor Kees Jalink and The Netherlands Cancer Institute (NKI) in Amsterdam. Having the meeting at a location where super-resolution microscopy is used on a daily basis makes a big difference, offering participants the chance to use live cells for workshops and see systems working in their true environments. Thanks also to the scientists that supported the meeting by coming and giving talks. As super-resolution continues to grow in importance in research, we recognize the need to come together to network, share information and experiences. Here we present the abstracts of the talks.Read article -
Detailed Morphological Characterisation of Hendra Virus Infection of Different Cell Types Using Super-Resolution and Conventional Imaging
Hendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. Our long-term aim is to understand the process of assembly of HeV virions. As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus encoded proteins in infected cells.Read article -
Universal PAINT – Dynamic Super-Resolution Microscopy
Super-resolution microscopy techniques have revolutionized biology for the last ten years. With their help cellular components can now be visualized at the size of a protein. Nevertheless, imaging living cells is a challenge for most of the super-resolution principles.Read article -
GSDIM Publication List
GSDIM microscopy is a widefield super-resolution technique based on the localization of fluorophores with nanometer precision. With its help a lateral resolution of down to 20 nm can be achieved, whereas the new 3D feature even shrinks axial resolution to 50 nm. Here we provide a collection of publications around that super-resolution microscopy method also called dSTORM.Read article -
Video Interview with Jean-Luc Vonesch
Jean-Luc Vonesch is head of the imaging facility at the Institute of Genetics and Molecular and Cellular Biology (IGBMC), Strasburg, France. 23 years ago he was the founder of this facility which nowadays serves more than 850 scientists distributed among 47 working groups. Looking deeply into the cells is of a special interest Vonesch states. And with super-resolution microscopy he pretends it is easier to identify the regions of interest for subsequent electron microscopy: “And so we can gain time thanks to the super-resolution” he says.Read article -
JC Virus Inclusions in Progressive Multifocal Leukoencephalopathy: Scaffolding Promyelocytic Leukemia Nuclear Bodies Grow With Cell Cycle Transition Through an S-to-G2–Like State in Enlarging Oligodendrocyte Nuclei
In progressive multifocal leukoencephalopathy, JC virus–infected oligodendroglia display 2 distinct patterns of intranuclear viral inclusions: full inclusions in which progeny virions are present throughout enlarged nuclei and dot-shaped inclusions in which virions are clustered in subnuclear domains termed “promyelocytic leukemia nuclear bodies” (PML-NBs). Promyelocytic leukemia nuclear bodies may serve a scaffolding role in viral progeny production.Read article -
Which Super-Resolution Method is Right for You?
Super-resolution microscopy has dramatically improved our understanding of intracellular dynamics, redefining what is possible in biological research. This infographic gives a compact overview on the different super-resolution techniques such as localization, structured illumination and stimulated emission depletion and will help you to choose the technology that best fulfills your individual research needs.Read article -
Video Interview with Rainer Pepperkok
Rainer Pepperkok is Head of the Advanced Light Microscopy Core Facility and Senior Scientist at the EMBL in Heidelberg (Germany). In the course of his studies he is interested in membrane traffic of the early secretory pathway in mammalian cells which he is trying to analyze with the help of most modern light microcopy techniques.Read article -
Super-Resolution Microscopy – Get Your Free e-Book for Download
Until recently, the diffraction of light had placed a fundamental limit on how far biologists could peer into cells with optical microscopes, preventing them from resolving features less than 250 nm in size, missing critical structures within cells. Over the past 20 years scientists have developed several ingenious techniques allowing them to resolve features as small as 20 nm.Read article -
Video Interview with Werner Zuschratter
Werner Zuschratter's personal focus is on analyzing the neuronal network, meaning the contacts between nerve cells. Out of this reason he started doing super-resolution microscopy: “It gives us deeper insight into the synapses, into the synaptic machinery, into the molecules we would like to see. Before we could only do electron microscopy and now, with super-resolution, we also have access by light microscopy to the deeper structures inside the nerve system.”Read article -
Video Interviews with Kees Jalink
Kees Jalink's group at the Netherlands Cancer Institute in Amsterdam, The Netherlands, explores signal transduction pathways and cell adhesion processes in cancer cells. In his eyes especially the new three-dimensional nanoscopic view of the relevant structure of interest is an essential feature to get the full picture.Read article -
Abstracts of the 4th European Super-Resolution User-Club Meeting
The 4th Super-Resolution User Club Meeting was held in collaboration with Christian Eggeling and the Weatherall Institute of Molecular Medicine in Oxford, UK. Here we present the abstracts of the talks and interviews with participants.Read article -
Video Interview with Dr. Yasushi Okada
Yasushi Okada, team leader at Riken Quantitative Biology Center in Osaka, Japan, investigates vesicular transport mechanisms in neuronal cells. As the size of transported vesicles is below 100 nm and the diameter of microtubules is about 25 nm, he uses super-resolution techniques to study the sophisticated machinery of neuronal transport.Read article -
Sample Preparation for GSDIM Localization Microscopy – Protocols and Tips
The widefield super-resolution technique GSDIM (Ground State Depletion followed by individual molecule return) is a localization microscopy technique that is capable of resolving details as small as 20 nanometers. GSDIM is suitable for a wide range of samples.Read article -
The Force of the Dark Side – Embedding Media for GSDIM Super-Resolution Localization Microscopy
Super-resolution microscopy such as Stimulated Emission Depletion (STED) and single-molecule based techniques rely on the same principle for breaking the diffraction limit: the unwanted fluorescence signals are switched off during the image acquisition process. Consequently, Ground State Depletion followed by Individual Molecule Return (GSDIM) microscopy and related techniques like PALM, STORM and dSTORM use metastable dark states of a fluorophore for temporal separation of single molecules.Read article -
Super-Resolution Microscopy Gives New Insights into Nuclear Pore Complex Organization
The Nuclear Pore Complex (NPC) is a large complex in the nuclear membrane, representing the gate to the eukaryotic genetic makeup. Because of this outstanding function the structure of the NPC is of great interest. Anna Szymborska, scientist at the EMBL in Heidelberg, comments on her resaerch results and the potential of Ground State Depletion microscopy (GSD) for protein complex analysis in the following interview.Read article -
Super-resolution Microscopy and the Third Dimension
Optical imaging devices have a finite depth of field and diffraction limited resolution. The depth of field problem was tackled first with confocal microscopes, diffraction unlimited resolution is available since a few years with super-resolution microscopes. Super-resolution microscopes with a solved depth of field problem are now available.Read article -
Three-Dimensional Super-Resolution GSDIM Microscopy
With the new 3D GSDIM technique structures like the Golgi and the microtubular network are resolved not only laterally, but also in a third dimension. The principle is based on the use of optical astigmatism to determine the accurate lateral and axial position of individual fluorochromes.Read article -
3D Localization Microscopy With Ground State Depletion (GSD)
With the latest development of a GSD 3D super-resolution platform, it is now possible to achieve a lateral resolution of down to 20 nm and an axial resolution of 70 nm. The technology is based on an astigmatism approach using a manipulated PSF to localize the molecule in z. This following tutorial describes the basic principles of the 3D GSD technology.Read article -
STED and GSDIM: Diffraction Unlimited Resolution for all Types of Fluorescence Imaging
This article gives an overview of two different types of superresolution techniques. Stimulated emission depletion (STED) microscopy is a versatile and fast method that is based on point scanning microscopy – usually an extension of a confocal microscope. Ground state depletion imaging (GSDIM) is a parallel recording widefield approach that explores inherent switching of fluorochromes and typically comes with a TIRF microscope. The two methods use very different approaches to reach the same goal: to see more details in light microscopes than possible when diffraction limited.Read article -
Tubulin Modifications Affect Monolayer Formation and Apical Trafficking in Epithelial Cells
The development and maintenance of polarized epithelial cells requires the establishment of complicated subcellular machinery. We studied the role of post-translational tubulin modifications within this process. At first, the distribution of detyrosinated microtubules was assessed in MDCK cells via immunofluorescence microscopy. No preferential accumulation of tyrosinated or detyrosinated microtubules could be detected at the apical or basal cell poles in epithelial cell cysts. However, during monolayer formation, the quantities of detyrosinated tubulin increased significantly over time.Read article
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