STED image of peripheral section of HeLa cell nucleus
STED image of peripheral section of HeLa cell nucleus

Abstracts of the 7th European Super-Resolution User-Club Meeting

Institute of Molecular Genetics of the ASCR, Prague, 24th - 26th October 2017

The 7th Super-Resolution User Club Meeting was held in collaboration with Prof Pavel Hozák, at the Institute of Molecular Genetics of the ASCR in Prague.

Keeping the event close to science is one of the founding principles of the event, allowing all participants to network, share and explore exciting new super-resolution and nanoscopy applications. Central to this are the scientific talks given during the meeting, with this cutting-edge microscopy technique as their central theme. A wide selection of topics were covered, prompting interesting discussions during the workshops. 

Here we present the abstracts of the talks held during the meeting. 

STED image of peripheral section of HeLa cell nucleus



Welcome to the 7th Super-Resolution User-Club Meeting

Prof. DrSc. Pavel Hozák, Head, Microscopy Centre, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague

A cell and molecular biologist, Pavel studied Biology at the Moscow State University, followed by research posts at the Dunn School of Pathology, Oxford University and at the Institute of Experimental Medicine of the Czechoslovak Academy of Sciences. Since 2006, he has been Head of the Department of Biology of the Cell Nucleus and since 2015, also Head of the Microscopy Centre, both at the Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic. He publishes in leading journals including Cell, Nature Cell Biology and Science. He lectures at the Charles University in the field of cellular and molecular biology, medical biology and microscopy. He is also the national coordinator of Czech-BioImaging and Euro-BioImaging.

Major research topics:

  • Definition of higher-order structures in the cell nucleus; mechanisms forming nuclear compartmentalization
  • Structure, dynamics, and function of the nucleoskeleton
  • Identification of nuclear structures active in epigenetic regulation of gene expression
  • Characterization of nuclear functions of myosin I, actin and actin-binding proteins
  • Localization and function of nuclear phospholipids in chromatin functions
  • Development of new methods in microscopy

In the Department of Cellular Biology Department of the Institute of Molecular Genetics of the ASCR, Prof. Hozák has long been involved in research related to the cell nucleus, particularly in the patterns governing genome functioning, because their disruption leads to an imbalance in gene activity and serious disease. Among the important discoveries of Prof. Hozák’s team, it has been found that the reading of genes is aided by myosin protein molecules and some lipids. His team also works towards improving electron microscopy methods that shift the possibilities of scientific research into cell structure. Prof. Hozak patented a set of nanoparticles of different shapes to make it possible to recognize the location of different molecules in cells, otherwise impossible with standard nanoparticles used.

Using super-resolution microscopy to determine the nuclear periphery organization

Dr. Jindřiška Fišerová, Department of Biology of the Cell Nucleus at the Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic

Nuclear periphery represents complex environment separating the nucleus and the cytoplasm. It is composed of double nuclear membrane with resident inner membrane proteins that is underlain with the nuclear lamina and perforated with nuclear pore complexes. In vertebrates, the nuclear lamina is formed by two lamin species – A- and B-type lamins that form separate but interconnected networks. Nuclear lamina is involved in mechanosensing of the cell, nuclear shape determination and strain resistance. Importantly, it functions in organizing chromatin and in turn, in associated processes including DNA replication, repair or translation. Whereas nuclear lamina is associated with rather gene-poor heterochromatin regions, NPCs are seen as islands of transcription activity and open chromatin.

We have used super resolution microscopy (SIM and STED) and subsequent image analysis to discern finer features of the organization of chromatin around nuclear pore complexes and the nuclear lamina. Further, we followed A- and B-type lamin organization in cells depleted of nuclear pore complex protein TPR and show that loss of large portion of nuclear pore complex baskets has wider implications in arrangement of chromatin at the nuclear periphery as well as in structural features of lamina networks.

Resolving cytoskeleton using multi-color STED and expansion microscopy

Dr. Eugene Katrukha, Senior scientist at the Biology Imaging Center at the University of Utrecht, the Netherlands

The internal space of a cell is filled with a set of scaffolding and structural polymer networks. Commonly referred to as a cytoskeleton, they provide railroads for the intracellular transport (microtubules), maintain cell shape (intermediate filaments) and drive cellular motility (actin filaments). The thickness of individual filaments is in the range of 8-20 nm, i.e. well below the diffraction limit. The talk will present examples of simultaneous configuration mapping for several of these networks using a set of superresolution techniques (STED, STORM, expansion microscopy and their combinations).

In a variety of cellular models (from fibroblasts to neurons and epithelial cells) these results provide insights into mechanisms of organelle positioning, transport organization, and internal pathological changes. I will show our best attempts, pitfalls and practical limits while pushing for multi-color, 3D, large field-of-view, live and “highest resolution possible” microscopy imaging.

Information in Localisation Microscopy

Dr. Susan Cox, Randall Division of Cell and Molecular Biophysics, King’s College London, UK

Localisation microscopy is a powerful tool for imaging structures at a lengthscale of tens of nm, but its utility for live cell imaging is limited by the time it takes to acquire the data needed for a super-resolution image. The acquisition time can be cut by more than two orders of magnitude by using advanced algorithms which can analyse dense data, trading off acquisition and processing time.

Modelling the entire localisation microscopy dataset using a Hidden Markov Model allows localisation information to be extracted from extremely dense datasets. This Bayesian analysis of blinking and bleaching (3B) is able to image dynamic processes in live cells at a timescale of a few seconds. The performance of 3B on various live cell systems is demonstrated, including cardiac myocytes and podosomes, showing a resolution of tens of nm with acquisition times down to a second.

While analysing higher density images can improve the speed at which the data required for a super-resolution reconstruction is acquired, there are still limits to the speed which can be achieved. Unlike in conventional fluorescence microscopy, these limits are not just set by the properties of the microscope, but are determined by the structure of the sample itself. This is because the local sample structure affects how quickly the information necessary to create an image of a certain resolution can be transmitted through the optical system. The theoretical limits will be discussed, and the effect on live cell experiments demonstrated.

Super-resolution microscopy: Challenges potentials and applications in biomedical research

Prof. Christian Eggeling, Professor of Molecular Immunology, MRC Human Immunology Unit & Scientific Director, Wolfson Imaging Centre Oxford, Weatherall Institute of Molecular Medicine, University of Oxford, UK

Understanding the complex interactions of molecular processes underlying the efficient functioning of the human body is one of the main objectives of biomedical research. Scientifically, it is important that the applied observation methods do not influence the biological system during observation. The most suitable tool that can cover all of this is optical far-field fluorescence microscopy. Yet, biomedical applications often demand coverage of a large range of spatial and temporal scales, and/or long acquisition times, which can so far not all be covered by a single microscope and puts some challenges on microscope infrastructure.

Taking immune cell responses and plasma membrane organization as examples, we outline these challenges but also give new insights into possible solutions and the potentials of these advanced microscopy techniques, e.g. for solving long-standing questions such as of lipid membrane rafts.

From Lamin to Lipids: STED as a tool in biomedical research

Prof. Marc van Zandvoort, Department of Molecular Cell Biology, Maastricht University, Maastricht, the Netherlands

The presentation will, after a short introduction into microscopy and STED, discuss some examples of 2D-STED on histological samples and microbubbles, to rapidly switch to a specific application, i.e., the imaging of Lamin structure around the cell nucleus of 3T3 cells.

Differences in resolution will be validated, demonstrating the gain of this technique in biological research. Possibilities for future research will be indicated.

Unveiling structural Super-Resolution information of human Amyloid-β (Aβ) plaques by combining STED microscopy and Array-Tomography

Dr. Jordi Andilla, Research Engineer, Super Resolution Light Microscopy and Nanoscopy Lab Facility,ICFO, Barcelona, Spain

Amyloid-β (Aβ) Plaques consist of aggregates of different species of Aβ peptides from monomers and oligomers to large fibrils. These plaques represent one of the core neuropathological traits of Alzheimer disease (AD). However, in human brain, studying these aggregates has been proven to be challenging. This is because the 3D information of the detailed structure that can be gathered from the sample is restricted in size by the diffraction limit of light. Furthermore, optical aberrations and scattering make it difficult to image planes inside the brain aggregates.

In this work, we present the combination of array tomography microscopy (ATM) and STED microscopy. This provides isotropic 3D information extraction with resolutions of up to 80nm without being compromised by the optical properties of the aggregates. We will see the applicability of this approach in the study of the aggregation of Aβ peptides in the AD and in the role of its oligomeric version (oAβ) which is believed to represent the most injurious species associated with synaptic toxicity and loss.

Live-cell STED using mNeonGreen

Dr. Martin Offterdinger, Head of the Biooptics Core Facility, Divison of Neurobiochemistry, Medical University of Innsbruck, Austria

Live cell STED microscopy using FPs is generally hampered by the fact that EGFP does not show a very good cross-section with many STED depletion lasers typically used for yellow-green chromophores at approx. 590 nm. EYFP and its derivatives show a much better cross-section at this wavelength but suffer from the fact that they tend to bleach rather easily. We have used mNeonGreen© in order to overcome these problems and found that is superior to both EGFP and EYFP possibly due to several specific properties. Firstly, it is clearly brighter than both EGFP and EYFP, secondly its fluorescence is red-shifted compared to EGFP thus yielding a much better cross-section with the depletion laser than EGFP. I will present several examples of successful live cell imaging using mNeonGreen in transfected cell lines.

Using super-resolution microscopy to elucidate the mitochondrial genome segretation machinery in Trypanosoma brucei

Prof. Dr. Torsten Ochsenreiter, Institute of Cell Biology, University of Bern, Switzerland

In almost all eukaryotes mitochondria maintain their own genome. Despite the discovery more than 50 years ago still very little is known about how the genome is properly segregated during cell division. The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure proper segregation of the mitochondrial genome via the basal bodies movement, during cell cycle.

Using super-resolution microscopy we precisely localize each of the currently known unique TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum towards the mitochondrial genome and that the assembly is not dependent on the kDNA itself.

Deciphering molecular control of deuterosome mediated centriole mass production

Dr. Camille Boutin, Developmental Biology Institute of Marseille, France

Cells bearing multiple motile cilia are found in a wide variety of metazoa, from marine invertebrates to humans. An early step of multiciliated cell (MCC) formation is the mass production of centrioles, which after conversion into basal bodies serve as anchoring units for cilia. Most centrioles in MCCs are produced from specialized structures called deuterosomes which measure few hundred nanometers in diameter.

40 years after its initial description, little is known about the composition, architecture and regulation of the deuterosome. I will introduce our strategy to identify deuterosome components and how STED microscopy provides me with a powerful tool to tackle its organization.

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