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

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.

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.

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.

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.

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.

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.

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.