The Super-resolution User Club, an initiative of Leica Microsystems, is intended to facilitate networking among users of a pioneering technology, create room to exchange ideas and experience and to stay ahead. The first part of this first meeting was dedicated to the exchange of experiences and results through talks, presentations and discussions about upcoming super-resolution techniques. The abstracts of the talks are shown below.
The second part of the meeting was organized as workshops, dedicated to STED and STED CW users. A GSD system was also available with dedicated time for demos and hands-on.
STED microscopy to study macromolecular architectures at synapses
Stephan Sigrist, Freie Universität Berlin, Institute for Biology, Germany
The majority of rapid cell-to-cell communication mechanisms and information processing within the nervous system makes use of chemical synapses. Thereby, the molecular organization of presynaptic active zones, the places where neurotransmitter filled synaptic vesicles get released, is a focus of intense investigation. We recently showed that Bruchpilot (BRP) of Drosophila melanogaster, which features homologies to the mammalian CAST/ERC family, is essential for structural organization and efficient neurotransmitter release at active zones (Kittel et al., 2006; Wagh et al., 2006).
Our group established protocols to directly visualize protein dynamics during synapse assembly and plasticity in living animals using confocal microscopy (Fuger et al., 2007; Rasse et al., 2005; Schmid et al., 2008). However, light microscopic inspection of synaptic organization is often restricted by the limited resolution of conventional light microcopy due to diffraction. Thus, we adapted a recent advance in high-resolution light microscopy (stimulated emission depletion microscopy, STED) for the analysis of synapse substructures. STED breaks the diffraction barrier and allows localization of proteins well below 100 nm. Using STED, we showed that BRP shapes the presynaptic active zone architecture by adopting an extended conformation, shining first light on the underlying macromolecular organization of active zones(Fouquet et al., 2009; Kittel et al., 2006; Owald et al., 2010). Now, we are combining STED sub-diffraction resolution with in vivo visualization of macromolecular organization.
- Fouquet W, Owald D, Wichmann C, Mertel S, Depner H, Dyba M, Hallermann S, Kittel RJ, Eimer S and Sigrist SJ: Maturation of active zone assembly by Drosophila Bruchpilot. J Cell Biol 186 (2009) 129-45.
- Kittel RJ, Wichmann C, Rasse TM, Fouquet W, Schmidt M, Schmid A, Wagh DA, Pawlu C, Kellner RR, Willig KI, Hell SW, Buchner E, Heckmann M and Sigrist SJ: Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release. Science 312 (2006) 1051–4.
- Owald D, Fouquet W, Schmidt M, Wichmann C, Mertel S, Depner H, Christiansen F, Zube C, Quentin C, Korner J, Urlaub H, Mechtler K and Sigrist SJ: A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila. J Cell Biol 188 (2010) 565–79.
- Rasse TM, Fouquet W, Schmid A, Kittel RJ, Mertel S, Sigrist CB, Schmidt M, Guzman A, Merino C, Qin G, Quentin C, Madeo FF, Heckmann M and Sigrist SJ: Glutamate receptor dynamics organizing synapse formation in vivo. Nat Neurosci 8 (2005) 898–905.
- Schmid A, Hallermann S, Kittel RJ, Khorramshahi O, Frolich AM, Quentin C, Rasse TM, Mertel S, Heckmann M and Sigrist SJ: Activity-dependent site-specific changes of glutamate receptor composition in vivo. Nat Neurosci 11 (2008) 659–66.
- Wagh DA, Rasse TM, Asan E, Hofbauer A, Schwenkert I, Durrbeck H, Buchner S, Dabauvalle MC, Schmidt M, Qin G, Wichmann C, Kittel R, Sigrist SJ and Buchner E: Bruchpilot, a protein with homology to ELKS/CAST, is required for structural integrity and function of synaptic active zones in Drosophila. Neuron 49 (2006) 833–44.
Alberto Diaspro, Italian Institute of Technology (IIT), Genoa, Italy
Two-photon excitation (2PE) fluorescence imaging cytometry is a powerful far-field optical approach for the study of the three-dimensional (3D) and dynamic properties of biological systems. The main advantages are given by better penetration in scattering samples and low overall phototoxicity/bleaching coupled to intrinsic 3D optical sectioning properties, while the main drawback lies in the loss of resolution and signal efficiency with respect to the 1PE case. For such reasons we decided to couple 2PE with STED microscopy (STimulated Emission Depletion). We aim to augment resolution and at the same time to improve the sample penetration capability of the STED approach. Results and related characterizations, obtained by means of 2PE STED-CW adapted architecture available at the Italian Institute of Technology, will be discussed.
- Bianchini P. and Diaspro A: J Microsc (OXF) 2011, in press.
- Mondal PP and Diaspro A: Scientific Reports (open access Nature Journal) 2011, in press.
- Dilipkumar S, Diaspro A, Mondal PP: Rev Sci Instrum 82:6 (2011) 063705.
Super-resolution investigation of synaptic vesicle recycling
Silvio Rizzoli, European Neuroscience Institute (ENI), Göttingen, Germany
Neurons communicate with each other by releasing neurotransmitter contained within small membrane-bound organelles termed synaptic vesicles. Synaptic vesicle function has been investigated over more than five decades, typicallythrough stimulation in vitro. This has led to the assumption that the vesicle role is to release neurotransmitter, and that synapses require numerous vesicles to sustain transmission during high activity. However, using live STED microscopy, among other microscopy assays, we have demonstrated that only a small population (pool) of vesicles releases neurotransmitter in vitro under physiological conditions. We followed this by an ethology approach, monitoring vesicle function in behaving animals (10 animal models, nematodes to mammals). Using FM dye photo-oxidation, pHluorin imaging, HRP uptake and electron microscopy, we found that only 1–5 % of the vesicles release neurotransmitter, even under the extreme stress of predation, or in synapses which fire constantly at high rates. All other vesicles (likely immobilized and inactivated by cross-linking proteins such as synapsin) serve as a buffer, binding soluble accessory proteins required for vesicle recycling, thus preventing their loss into the axon. Calcium controls the buffering process, ensuring that soluble proteins are delivered upon demand. We conclude that the functional organization of neurons can be accurately interpreted only under physiological (in vivo) conditions.
- Denker A, Bethani I, Kröhnert K, Körber C, Horstmann H, Wilhelm BG, Barysch SV, Kuner T, Neher E, Rizzoli SO: A small pool of vesicles maintains synaptic activity in vivo. Proc Natl Acad Sci USA Sep 8 (2011) [Epub ahead of print].
- Denker A, Kröhnert K, Bückers J, Neher E, Rizzoli SO: The reserve pool of synaptic vesicles acts as a buffer for proteins involved in synaptic vesicle recycling. Proc Natl Acad Sci USA Sep 8 (2011) [Epub ahead of print].
- Hoopmann P, Punge A, Barysch SV, Westphal V, Bückers J, Opazo F, Bethani I, Lauterbach MA, Hell SW, Rizzoli SO: Endosomal sorting of readily releasable synaptic vesicles. Proc Natl Acad Sci USA Nov 2 107:44 (2010) 19055–60. Epub 2010 Oct 18.
- Kamin D, Lauterbach MA, Westphal V, Keller J, Schönle A, Hell SW, Rizzoli SO: High- and low-mobility stages in the synaptic vesicle cycle. Biophys J Jul 21 99:2 (2010) 675–84.
Live Cell STED Microscopy
Katrin Willig, Max Planck Institute for Biophysical Chemistry, Department of NanoBiophotonics, Göttingen, Germany
The green fluorescent protein (GFP) and its derivatives have revolutionized the imaging of living cells by providing specific labeling of proteins through genetic fusion. Over the past decade innovative techniques have been developed, which can also attach chemically synthesized fluorophores to proteins within living cells. Most of these labeling techniques are compatible with STED microscopy and can be used to acquire super-resolution images in living cells. This will be shown using various examples in this talk.
The yellow fluorescent protein variant Citrine was the first to be used with STED microscopy to image structures inside of a living cell . With Citrine targeted at the endoplasmic reticulum (ER) STED images were recorded with sub-diffraction resolution of <50 nm (Figure 1). Time-lapse STED recordings documented morphological changes of the ER over time. Also, GFP fusion proteins were imaged in intact living organisms, namely Caenorhabditis elegans nematodes [2, 3]. Bimolecular fluorescence complementation (BiFC) was used to image protein colocalization with sub-diffraction resolution . To achieve this, a nonfluorescent fragment of the yellow fluorescent protein Citrine was fused to tubulin and its counterpart to the microtubulin-associated protein MAP2. As soon as the fragment-carrying proteins come into contact, the fluorescence of the re-formed Citrine is reconstituted. STED images with resolutions of up to 65 nm prove to be a powerful tool for studying protein colocalization in living cells at the nanoscale.
Different techniques are commercially to label living cells with organic dyes. The so called "SNAP-tag" relies on human O6-alkylguanine-DNA alkyltransferase (hAGT) as a tag, which can be fused to a host protein. This tag has a size of 182 amino acids (aa), so it is slightly smaller than fluorescent proteins (~240 aa). We used tetramethylrhodamine (TMR) to image the cytoskeleton as well as structures located at the cell membrane (caveolin and connexin-43) with a resolution down to 40 nm (Figure 2) .
Fig. 1: Sub-diffraction-resolution imaging of the ER in a living mammalian cell1. Shown are confocal and simultaneously recorded STED (x, y) images from the ER labeled with the fluorescent protein Citrine revealing features of 52 nm FWHM as indicated by the profile (raw data: 35.7 mW STED focal intensity), indicating that the lateral resolution in the STED image is <50 nm. Scale bar = 500 nm.
Even though STED microscopy has been extended for use with multiple colors in various different settings, two-color measurements in living cells have been hampered by the complexity involved. In a technically simple approach photochromic or reversible switchable fluorescent proteins (RSFPs) can be used, which are genetically encoded and hence are inherently live-cell compatible. They may be reversibly photoswitched from a non-fluorescent (off) to a fluorescent (on) state and back again by irradiation with light of different wavelengths. Here, the longer switching wavelength also leads to fluorescence emission. RSFPs exhibit either a negative switching mode, i.e. the light eliciting fluorescence induces the off-switching, or a positive switching mode in which the on-switching is induced. We use both the positive switching RSFP Padron and the negative switching Dronpa-M159T, which both feature a similar excitation and emission spectrum and therefore can function utilizing a single STED wavelength (Figure 3) . Because of the antipodal switching characteristics these RSFPs can alternately be switched to the on-state and then sequentially recorded by STED nanoscopy. This approach requires adding just a single wavelength to perform RSFP switching.
- Hein B, Willig KI, Hell SW: Stimulated emission depletion (sted) nanoscopy of a fluorescent protein-labeled organelle inside a living cell. Proceedings of the National Academy of Sciences of the United States of America 105:38 (2008) 14271–76.
- Willig KI, Kellner RR, Medda R, Hein B, Jakobs S, Hell SW: Nanoscale resolution in GFP-based microscopy. Nature Methods 3:9 2006) 721–723.
- Rankin BR, Moneron G, Wurm CA, Nelson JC, Walter A, Schwarzer D, Schroeder J, Colón-Ramos DA, Hell SW: Nanoscopy in a Living Multicellular Organism Expressing GFP. Biophysical Journal 100:12 (2011) L63– L65.
- Lalkens B, Testa I, Willig KI, Hell SW: MRT letter: Nanoscopy of protein colocalization in living cells by STED and GSDIM. Microscopy Research and Technique n/a (2011) n/a.
- Hein B, Willig KI, Wurm CA, Westphal V, Jakobs S, Hell SW: Stimulated Emission Depletion Nanoscopy of Living Cells Using SNAP-Tag Fusion Proteins. Biophysical Journal 98 (2010) 158–163.
- Willig KI, Stiel AC, Brakemann T, Jakobs S, Hell SW: Dual-Label STED Nanoscopy of Living Cells Using Photochromism. Nano Letters n/a (2011) n/a.
Novel developments of STED: fluorescence correlation spectroscopy and gated CW STED
Christian Eggeling, Max-Planck-Institute for Biophysical Chemistry, Department of NanoBiophotonics, Göttingen, Germany
Stimulated Emission Depletion (STED) far-field microscopy allows the study of living cells with nanoscale resolution, otherwise impeded by the limited spatial resolution of conventional microscopes . Besides the recording of images, the combination of STED with single-molecule sensitive spectroscopic tools such as Fluorescence Correlation Spectroscopy (FCS) discloses complex dynamical processes hidden to the conventional observations. On one hand, STED realizes the application of FCS to systems where molecular concentrations of several micro-mol per liter (µM) are required [2-4]. On the other hand, STED-FCS offers novel insights into important cellular processes, such as lipid-lipid, lipid-protein interactions or the formation of so-called "lipid-rafts" in the cellular plasma membrane [4–7].
The performance of STED(-FCS) nanoscopy can be further improved exploiting spectroscopic information of the emitted fluorescence signal such as the lifetime of the excited state, which is quenched upon stimulated emission. Consequently, combining STED nanoscopy with gated detection facilitates tuning of the spatial resolution and enables nanoscale imaging and FCS with rather low continuous-wave laser powers .
- Hell SW: Far-Field Optical Nanoscopy. Science 316 (2007) 1153.
- Kastrup L et al.: Fluorescence Fluctuation Spectroscopy in Subdiffraction Focal Volumes. PRL 94 (2005) 178104.
- Blom H et al.: Fluorescence Fluctuation Spectroscopy in Reduced Detection Volumes. Curr Pharm Biotechnol 7 (2006) 51.
- Ringemann C et al.: Exploring single-molecule dynamics with fluorescence nanoscopy. New J Physics 11 (2009) 103054.
- Eggeling C et al.: Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457 (2009) 1159.
- Polyakova SM et al.: New GM1 Ganglioside Derivatives for Selective Single and Double Labelling of the Natural Glycosphingolipid Skeleton. Eur J Org Chem 5162 (2009).
- Kolmakov K et al.: Red-Emitting Rhodamine Dyes for Fluorescence Microscopy and Nanoscopy. Chem Eur J 16 (2010) 158.
- Vicidomini G et al.: Sharper low power STED nanoscopy by time gating. Nat Meth. (2011); doi:10.1038/nmeth.162.
Focus on synapses: live-cell super-resolution STED imaging of brain slices
Valentin Nägerl, Université Bordeaux Segalen, Synaptic Plasticity and Superresolution Microscopy Group, Institute for Interdisciplinary Neuroscience, France
Neuronal synapses are composed of a pre- and a postsynaptic membrane specialization, forming elementary functional compartments for rapid and flexible signaling in the central nervous system. Understanding how synapses are built during development and modified by experience is a central theme for neuroscience.
However, as they are typically very small (<1 mm) and dynamic and reside inside three-dimensional, light-scattering tissue, it is difficult to study them by conventional, diffraction-limited light microscopy.
However, major advances in superresolution imaging and fluorescence labeling are greatly improving our ability to investigate the inner life and dynamics of synapses using life-cell imaging approaches. We have previously shown that superresolution STED microscopy is a powerful technique for live-cell imaging of synapse morphology using YFP as a genetically encoded volume-label.
We will review our recent progress in adapting STED microscopy for live-cell nanoscale imaging deep inside biological tissue and in two colors simultaneously. Specifically, we will demonstrate the powerful potential of these methodological advances for several applications concerning superresolution imaging of synapses: 1) spine plasticity and actin dynamics using lifeact; 2) nanoscale imaging up to 120 mm deep below tissue surface and 3) dual-color, live-cell imaging of pre- and postsynaptic structures with nanoscale spatial resolution using spectral unmixing of GFP and YFP and other popular green fluorescent dyes.
Fig. 2: Confocal (left) versus STED (right). Nuclear pore complexes and clathrin coated vesicles of adherent cell. Labels: BD Horizon V500 (red) and Oregon Green 488 (green) Super-resolution in two colors is essential to put biological information into context and allow co-localization studies at the nanoscale. Combination of a large Stokes shift dye (e.g. BD Horizon V500) together with a standard fluorophore (e.g. Oregon green 488) allows to obtain super-resolution in two channels applying only one STED wavelength (592 nm). The differences in the excitation and emission spectra are exploited to distinguish the two dyes in sequential recordings. The advantage of applying the same STED doughnut for both super-resolution channels keeps the set up simple and also avoids chromatic aberration problems, which other implementations of super-resolution microscopy need to compensate. Courtesy of MPI for Biophysical Chemistry, Dept. of Nanobiophotonics, Goettingen, Germany.