Abstracts of the 4th European Super-Resolution User-Club Meeting

Abstracts of the talks & interviews with participants

August 08, 2014

The 4th Super-Resolution User Club Meeting was held in collaboration with Christian Eggeling and the Weatherall Institute of Molecular Medicine in Oxford, UK.

“Once again this meeting was a great success as a networking opportunity and a forum for users to meet and discuss super-resolution applications and challenges, and as an opportunity for Leica Microsystems to ‘touch base’ with its growing super-resolution community and gather valuable feedback,” says Joanne Fallowfield, Marketing Manager Life Science Research EMEA at Leica Microsystems. “We had excellent speakers, who shared with us their experience on applying super-resolution or other advanced fluorescence techniques to their research.”

 

 


Talk abstract: Insights into plasma membrane organization using STED microscopy

Christian Eggeling

Christian Eggeling, University of Oxford, Weatherall Institute of Molecular Medicine, Oxford, UK

Lipid-lipid and lipid-protein plasma membrane interactions such as the formation of lipid nanodomains (often denoted "rafts") or restrictions of molecular plasmamembrane diffusion by cortical cytoskeleton compartments are considered to play a functional part in a whole range of membrane associated processes. However, the direct and non-invasive observation of such structures in living cells is impeded by the resolution limit of >200 nm of a conventional far-field optical microscope. With the superior spatial resolution of STED nanoscopy with effective focal spot sizes down to 20–30 nm in living cells, it is now possible to directly resolve such membrane heterogeneities, e.g. by imaging and investigating protein nanoclusters on the membrane surface [1]. On the other hand, the combination of STED nanoscopy with tools such as fluorescence correlation spectroscopy (FCS) allows the disclosure of complex nanoscopic dynamical processes. By performing FCS measurements in focal spots tuned to a diameter of down to 30 nm, we have obtained new details of molecular membrane dynamics. Unlike fluorescent phosphoglycerolipids, fluorescent sphingolipids or a number of proteins are transiently (~10 ms) trapped on the nanoscale in often cholesterol-mediated molecular complexes [2]. These interactions are distinct for different lipids and proteins and may play an important role in cellular functionality [3]. Comparison of these trapping characteristics to the organization and dynamics of the different fluorescent lipid analogues in model membranes reveals details of the role of lipid “rafts” [4]. On the other hand, the comparison of lipid and protein dynamics in different cells reveals different diffusion modes, mainly depending on the organization of the underlying (actin) cytoskeleton. Furthermore, improved insights into membranes heterogeneities are realized by recent technological developments of the STED FCS approach [5]. The novel observations shed new light on the role of lipid-protein interactions and nanodomains for membrane bioactivity in a whole gamut of cellular processes. Examples will be given for molecular processes of immune cells following infection.

References

  1. Donnert G, Keller J, Medda R, Andrei MA, Rizzoli SO, Lührmann R, Jahn R, Eggeling C, and Hell SW: Macromolecular-scale resolution in biological fluorescence microscopy. PNAS 103 (31): 11440–45 (2006); doi: 10.1073/pnas.0604965103 (2006).
    Sieber JJ, Willig KI, Kutzner C, Gerding-Reimers C, Harke B, Donnert G, Rammner B, Eggeling C, Hell SW, Grubmüller H, and Lang T: Anatomy and Dynamics of a Supramolecular Membrane Protein Cluster. Science 317 (5841): 1072–76 (2007); doi: 10.1126/science.1141727.
  2. Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S, Belov VN, Hein B, von Middendorff C, Schönle A, and Hell SW: Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457: 1159–62 (2009); doi: 10.1038/nature07596.
    Ringemann C, Harke B, von Middendorff C, Medda R, Honigmann A, Wagner R, Leutenegger M, Schönle A, Hell SW, and Eggeling C: Exploring single-molecule dynamics with fluorescence nanoscopy.New J. Phys. 11: 103054 (2009); doi: 10.1088/1367-2630/11/10/103054.
    Vicidomini G, Moneron G, Han KY, Westphal V, Ta H, Reuss M, Engelhardt J, Eggeling C, and Hell SW: Sharper low-power STED nanoscopy by time gating. Nature Methods 8: 571–73 (2011); doi: 10.1038/nmeth.1624.
  3. Mueller V, Ringemann C, Honigmann A, Schwarzmann G, Medda R, Leutenegger M, Polyakova S, Belov VN, Hell SW, and Eggeling C: STED Nanoscopy Reveals Molecular Details of Cholesterol- and Cytoskeleton-Modulated Lipid Interactions in Living Cells. Biophys J. 101 (7): 1651–60 (2011); doi: 10.1016/j.bpj.2011.09.006.
  4. Sezgin E, Levental Y, Grzybek M, Schwarzmann G, Mueller V, Honigmann A, Belov VN, Eggeling C, Coskun Ü, Simons K, and Schwille P: Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes. Biochimica et Biophysica Acta (BBA) – Biomembranes 1818 (7): 1777–84 (2012).
  5. Honigmann A et al.: Faraday Discuss. (2012); doi: 10.1039/C2FD20107K.
    Vicidomini G, Moneron G, Han KY, Westphal V, Ta H, Reuss M, Engelhardt J, Eggeling C, and Hell SW: Sharper low-power STED nanoscopy by time gating. Nature Methods 8: 571–73 (2011); doi: 10.1038/nmeth.1624.
  6. Mueller V, Eggeling C, Karlsson H, and Von Gegerfelt D: CW DPSS Lasers Make STED Microscopy More Practical. BioPhotonics 19: 20 (2012).

Interview with Christian Eggeling

Dr. Christian Eggeling, group leader at the MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine (WIMM), University of Oxford, and scientific head of Wolfson Imaging Centre Oxford at WIMM, UK:


Talk abstract: Biological applications for 3D STED microscope systems with two depletion lasers

Timo Zimmermann

Timo Zimmermann, Centre for Genomic Regulation, Advanced Light Microscopy Unit, Barcelona, Spain

Stimulated Emission Depletion (STED) constitutes a powerful method for resolution improvement beyond the diffraction limit in light microscopy. Additional time-gating (gSTED) of the detected fluorescence signal in systems with continuous wave (CW) laser depletion improves image resolution even further.

Until recently, these improvements were limited in gated CW-STED systems to the lateral resolution of the image and mainly to the depletion at 592 nm of fluorophores that emit in the green range of the spectrum.

The recently introduced possibility to flexibly improve axial resolution in STED imaging and the introduction of an additional laser line for depletion at 660 nm offer new ways to image biological samples. We have taken highly resolved 3D stacks and multichannel datasets of a variety of specimens and structures to make full use of these new features for several projects using the improved z-resolution and orange to red dyes and dye combinations for the new depletion laser line.

The requirements to take a high number of z-sections and the role of deconvolution for the processing of STED datasets will be discussed. Also, examples of multichannel measurements using 592 nm only, 660 nm only and also a combination of the two wavelengths for depletion will be given.

Fig.: Confocal and 3D STED image of a marine protozoan (Dinoflagellata) using 200 optical sections. The tubulin cytoskeleton was labeled with an α-tubulin antibody and Oregon Green 488. Displayed are the frontal and sideways projections of the confocal dataset, of the STED raw data and the deconvolved STED data.

Talk abstract: In vivo STED microscopy

Katrin Willig

Katrin Willig, University of Göttingen, Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany

Confocal and two-photon microscopy are powerful techniques for imaging structures inside living cells, tissue or living animals. However, they cannot show fine details or substructures of the cell because of their diffraction-limited resolution of about half of the wavelength of light (~200–350 nm). From all the super-resolution microscopy techniques presently available, STED microscopy stands out for its imaging capabilities in tissue: It is live-cell compatible, especially when using standard fluorescent proteins, it is able to record 3D images from inside transparent tissue and the imaging speed is fast compared to other super-resolution methods.

Here we present an application of STED microscopy to image structures in the brain of a living mouse. Synapses, which are the contact sides between neurons, are the most fundamental information processing units in the brain. Excitatory synapses are mostly formed on small dendritic protrusions, the dendritic spines. The minute size of synapses makes them an ideal target for STED microscopy. It was shown that STED microscopy is capable of imaging dendritic spines up to 120 μm deep inside living organotypic brain slices and resolving distinct distributions of actin inside dendrites and spines [1]. Now we have developed an upright STED microscope to image the cerebral cortex of a living mouse through a glass window, so that we could observe the dynamics of dendritic spines in the molecular layer of the visual cortex [2]. We revealed filamentous actin (Figure 1) in dendritic spines and the dendritic arborization down to a depth of 40 μm [3]. Time-lapse recordings revealed dynamic changes at a resolution of ~60 nm.

These results show that STED nanoscopy is a highly suitable tool for neuroscience which can play a substantial role in the study of the living brain. These results show that STED nanoscopy is a highly suitable tool for neuroscience which can play a substantial role in the study of the living brain.

Fig.: In vivo STED microscopy of dendritic F-actin in the molecular layer of the visual cortex. Maximum intensity projection of a stack of 5 (xy) STED images taken every 500 nm axial (z) distance. Right panels: Line profile of the marked positions; average of 3 lines (green) or 5 lines (blue) of the raw data and Lorentz fit with full-width at half-maximum (FWHM); all data are raw. Adapted from Willig et al.


Talk abstract: Applying SR GSD to study the cytoskeleton: challenges, solutions, results, and new challenges

Kees Jalink

Nahidi Azar L, Leyton-Puig D, van den Broek B, Klarenbeek J, Manders E, and Jalink K, van Leeuwenhoek Centre for Advanced Microscopy, and Netherlands Cancer Institute, Amsterdam, The Netherlands

For centuries, the light microscope has played a pivotal role in biology and medicine. Without the ability to observe cells and tissues, to study their morphology, dynamics and molecular composition, we would have only a fraction of the understanding of life we have today. Yet, until recently, we failed to have the resolution to observe the building blocks of life, as the resolution of light microscopy is about 50-fold too low. Super-resolution microscopy is rapidly changing that. We use Ground State Depletion Imaging to study the composition of the cytoskeleton and to decipher signal transduction mechanisms.

As a first step, we have invested in optimizing preparation and recording conditions. Blinking conditions have been optimized and algorithms were developed for drift correction and background subtraction. We are currently applying SR GSD in a number of biological studies. In collaboration with the Sonnenberg lab in our institute, we have focussed on the structure of hemidesmosomes, adhesive structures within the skin. We also study keratin filament formation and plasticity in collaboration with Dr. Reinhard Windhoffer in Aachen, Germany. In my talk, I discuss image optimization, provide examples of new insights that were obtained with the system, and also discuss‚ new problems that pop up due to super-resolution.


Interview with Leila Nahidi Azar

Leila Nahidi Azar, Ph.D. Student, Department of Cell Biology, The Netherlands Cancer Institute (NKI-AVL), Amsterdam (Kees Jalink group):


Talk abstract: From 2D to 3D Super-resolution microscopy: Examples of current research conducted at the Advanced Light Microscopy Facility of the EMBL Heidelberg

Marko Lampe

Marko Lampe, EMBL, Advanced Light Microscopy Facility, Heidelberg, Germany

Super-resolution fluorescence microscopy has the potential to deliver new insight into cellular and structural biology. The implementation of GSDIM and STED Super-resolution microscopy into the EMBL Advanced Light Microscopy facility (ALMF), the interplay with established technologies and current examples of scientific projects will be presented in this talk. In 2011, the Leica SR GSD using GSDIM/dSTORM was installed in the ALMF and successfully applied to study the structure of nuclear pores by Anna Szymborska in Jan Ellenberg’s Lab [1]. The facility introduced 3D SR imaging to their STED and GSDIM systems in 2013, which not only increased the potential of the microscopes but also the requirements for precise sample preparation. Therefore, current sample preparation procedures are briefly presented and compared: in the context of the typical imaging workflow in the facility and also as a factor that can cause optical aberrations.

The choice of the appropriate technology – STED or GSDIM – along its respective advantages and limitations is crucial for the subsequent success of a project and will be discussed along recent examples from users and members of the imaging facility.


Talk Abstract: Beam generation and aberration control in STED microscopy using spatial light modulators

Mark Neil

Mark Neil, Imperial College London, Department of Physics, Faculty of Natural Sciences, Photonics Group, London, UK

The performance of STED microscopes is highly dependent on the shape and fidelity of the depletion beam that suppresses the outer parts of the excitation point spread function in order to allow super-resolved imaging. Fixed phase masks can be used to help shape the depletion beam, but are unable to accommodate changes in its shape either to vary the mode of operation or as a result of aberrations. Spatial light modulators (SLM) offer the ability to dynamically alter the pupil plane phase of the depletion beam under direct computer control and hence are a flexible and powerful alternative to fixed phase masks.

A beam generation system is presented based on a liquid crystal SLM where both lateral and axial resolution enhancement can be achieved simultaneously by multiplexing appropriate depletion beams onto incoherent orthogonal polarisation states. Though the SLM is a continuous phase device, further accuracy is achieved by displaying holograms in order to generate the appropriate phase modulation. In addition the holograms can be programmed to account for optical aberrations in the microscope, either introduced by the system itself or within the sample.

Results are presented using the microscope to image the immune synapse that forms when natural killer cells check other cells for infection or dysfunction. In a conventional slide preparation these synapses tend to form in a plane oriented parallel to the microscope axis and so being able to achieve both lateral and axial resolution enhancement is important for imaging structures within the synapse.

In another application, nitrogen-vacancy colour centres are imaged as part of a study into the differences between synthetic and natural diamonds. Here the high refractive index of diamond introduces large amounts of spherical aberration when focusing even only a few microns below the surface. We show that the resulting distortions of the depletion beams can be corrected and that resolution enhancement can be restored.


Interview with Guiseppe Vicidomini

Guiseppe Vicidomini, Ph.D., Department of Nanophysics, Italian Institute of technology (IIT), Genoa: 


Talk abstract: Two quite versatile fluorescent proteins

Sergi Padilla-Parra

Sergi Padilla-Parra, University of Oxford, Wellcome Trust Centre for Human Genetics, Division of Structural Biology, Oxford, UK

Choosing the right fluorescent protein (FP) is crucial when designing quantitative microscopy experiments in live cells. Different approaches require different FP’s with particular and well characterized physic-chemical characteristics. Picking and cloning different FP’s adapted to fulfil the needs of specific microscopy techniques can be time consuming and tedious. Recent advances in FP engineering allow scientists to choose among a big variety of FP’s, all of them with their pros and cons. Since the 488 and 561 nm laser lines are a common choice in almost all microscopes and techniques we reasoned that a particular green/red FP pair could be flexible enough for many approaches in quantitative fluorescence imaging microscopy in the context of live cells. After analysing many green/red FPs we found that the monomeric version of Clover (mClover) and mRuby are good candidates to be used quantitatively in many microscopy techniques. Light induced dark states (triplet state), intrinsic dark states, photostability, single exponential behavior of their fluorescence decay and high brightness make this couple a very attractive and flexible choice for FRET-FLIM, FCCS, dual color TIRF microscopy and dSTORM super-resolution in live cells.


Interview with Marc van Zandvoort

Marc van Zandvoort, Ph.D., leader of the multiphoton groups, both at Maastricht University and RWTH Aachen; Vice-President and Secretary of the Vital Imaging Steering Committee of Maastricht University, The Netherlands.

Comments

Help us to improve the Leica Science Lab and participate in our survey! Start Survey

Arrow Down

Arrow Up