"People really interact during the sessions – this is really important and helps to create a nice atmosphere. I look forward to participating in the 3rd meeting", said Giuseppe Vicidomini, one of the speakers at this year's meeting, who specializes in gated confocal super-resolution techniques at the IIT, Genoa, Italy.
"The meeting enables users of Leica Microsystems ground-breaking super-resolution systems to meet and share experiences – we are happy to provide a platform for our customers to do that, and to continue to support the work they do in research science" says Baba Awopetu, European Marketing Director, Leica Microsystems.
Science for Life Laboratory – a Swedish national resource center for molecular biosciences and medicine
Hjalmar Brismar, Science for Life Laboratory, Karolinska Institute, Stockholm, Sweden
The Science for Life Laboratory (SciLifeLab) is a national infrastructure for large-scale biological and medical research with a focus on genomics, proteomics, bioimaging and bioinformatics. SciLifeLab was created by a coordinated effort from four universities in Stockholm and Uppsala and was inaugurated in 2010. The center combines advanced technical know-how and state-of-the-art equipment with a broad knowledgebase in translational medicine and technology-driven molecular bioscience.
The vision is to make SciLifeLab a competitive center for high-throughput bioscience – focusing on large-scale DNA sequencing, expression analysis, protein profiling, cellular profiling, bioimaging, advanced bioinformatics and systems biology . The SciLifeLab initiative spans four universities and two sites, one in Stockholm and one in Uppsala. It has been made possible by strategic research grants from the Swedish government.
The Stockholm site, including the Karolinska Institutet, the KTH Royal Institute of Technology and the Stockholm University, is built around four technical platforms; genomics, proteomics, bioinformatics and bioimaging. The laboratory is located to the Karolinska Institutet Campus. Currently approximately 300 researchers are active in the Stockholm site. During 2013 more space is made available and an additional 350 researchers will move into the center.
The genomics platform is based on high capacity next-generation DNA sequencing and has a throughput equal to several hundreds of complete human genomes per year. The proteomics platform includes facilities for mass spectrometry, antibody-based protein analysis and automated screening with chemical libraries and siRNA technologies.
The genomics and proteomics platforms are complemented by the bioimaging platform with facilities for advanced light microscopy, including STED and other types of super-resolution microscopy. A strategic collaboration has been established between SciLifeLab and the Human Protein Atlas project 2, providing one of the world’s largest collections of antibodies against human proteins (at present against 50 % of the human proteins). SciLifeLab is using these antibodies in a number of large-scale projects, e.g. in mapping the subcellular localization of human proteins.
- Travis: Sweden bets on new lab to spruce up its bioscience future. Science 328 (2010) 805.
- Uhlen et al: Towards a knowledge-based Human Protein Atlas. Nature Biotechnology 28:12 (2010) 1248–50.
Nanoscopy with focused light
Stefan W. Hell, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
In STED microscopy , fluorescent features are switched off by the STED beam, which confines the fluorophores to the ground state everywhere in the focal region except at a subdiffraction area of extent
d ≈ λ/(2 NA√[I + Is]).
In RESOLFT microscopy [2, 3], the principles of STED have been expanded to fluorescence on-off-switching at low intensities I, by resorting to molecular switching mechanisms that entail low switching thresholds Is. An Is lower by many orders of magnitude is provided by reversibly switching the fluorophore to a long-lived dark (triplet) state [2–4] or between a long-lived "fluorescence activated" and "deactivated" state [2, 5]. These alternative switching mechanisms entail an Is that is several orders of magnitude lower than in STED. In imaging applications, STED/RESOLFT enables fast recordings and the application to living cells, tissues, and even living animals [6, 7].
Starting from the basic principles of nanoscopy we will discuss recent developments [8, 9] with particular attention to RESOLFT and the recent nanoscale imaging of the brain of living mice  by STED.
- Hell SW, Wichmann J: Breaking the diffraction resolution limit by stimulated-emission – stimulated-emission-depletion fluorescence microscopy. Opt Lett 19 (1994) 780–782, doi:10.1364/OL.19.000780.
- Hell SW: Toward fluorescence nanoscopy. Nat Biotechnol 21 (2003) 1347–1355.
- Hell SW, Jakobs S, Kastrup L: Imaging and writing at the nanoscale with focused visible light through saturable optical transitions. Appl Phys A 77 (2003) 859–860.
- Hell SW: Far-Field Optical Nanoscopy. Science 316 (2007) 1153–1158.
- Hofmann M, Eggeling C, Jakobs S, Hell SW: Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. PNAS 102 (2005) 17565–17569.
- Rankin BR et al: Nanoscopy in a Living Multicellular Organism Expressing GFP. Biophys J 100 (2011) L63–L65.
- Berning S, Willig KI, Steffens H, Dibaj P, Hell SW: Nanoscopy in a Living Mouse Brain. Science 335 (2012) 551.
- Grotjohann T et al: Diffraction-unlimited all-optical imaging and writing with a photochromic GFP. Nature 478 (2011) 204–208.
- Brakemann T et al: A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching. Nat Biotechnol 29 (2011) 942–947.
Single-molecule studies on a STED microscope: New insight into membrane organization
Christian Eggeling, Veronika Mueller, Alf Honigmann, Giuseppe Vicidomini, Gael Moneron, Haisen Ta, Stefan W. Hell
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 [2–4]. For example, STED-FCS offers novel insights into important cellular processes, such as lipid-lipid, lipidprotein interactions or the formation of so-called "lipid-rafts" in the cellular plasma membrane [4–9]. Improved insights are realized by the implementation of gated detection or by recording STED-FCS data during scanning [10–12].
- 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.
- Mueller V et al: STED Nanoscopy Reveals Molecular Details of Cholesterol- and Cytoskeleton-Modulated Lipid Interactions in Living Cells. Biophys J 101 (2011) 1651.
- Honigmann A et al: STED microscopy detects and quantifies liquid phase separation in lipid membranes using a new far-red emitting fluorescent phosphoglycerolipid analogue. Faraday Discussion, doi: 10.1039/C2FD20107K (2012).
- Vicidomini G et al: Sharper low power STED nanoscopy by time gating. Nat Meth 8 (2011) 571.
- Honigmann A et al: Super-resolution STED microscopy advances with yellow CW OPSL. Laser Focus World 48 (2012) 75.
- Mueller V et al: CW DPSS Lasers Make STED Microscopy More Practical. Biophotonics 19 (2012) 30.
Radial organization of the nuclear pore complex imaged by super-resolution microscopy
Anna Szymborska, Alex de Marco, Volker Cordes, John Briggs and Jan Ellenberg; EMBL, Cell Biology and Biophysics, Heidelberg, Germany
Integration of atomic structures of individual proteins into macromolecular models of multiprotein assemblies is a long standing challenge in the understanding of the mechanistic details and function of cellular machinery. One of the most prominent examples is the nuclear pore complex (NPC), a ~100 MDa assembly of several hundred polypeptides that functions as the sole channel for nucleocytoplasmic transport in all eukaryotes. Cryo-electron tomography has provided an overall structure of NPC, but due to technical limitations its resolution does not allow to unambiguously map the positions, stoichiometry and orientation of individual subcomplexes or atomic structures of single nucleoporins within the assembly. How the individual molecular building blocks assemble to make the nuclear pore therefore remains an open question.
Here, we have used super-resolution (SR) light microscopy to study the substructure of the NPC in human cultured cells. We initially focused on the organization of the Nup107–160 complex, which is the major component of the vertebrate NPC’s scaffold and is responsible for stabilization of the highly curved pore membrane. We overcame the typical resolution limit of SR in biological specimen of ~20 nm by combining it with single particle averaging of thousands of pores, and measured the distances of Nup107–160 components from the central 8-fold symmetry axis of the pore with 1 nm precision. We obtained new structural insight into the organization of the NPC scaffold and demonstrate that structural biology questions can be addressed by light microscopy.
Talking about Super-resolution: variations on the theme
Alberto Diaspro, Department of Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy & Department of Physics, Università degli Studi di Genova, Genoa, Italy
It is well known and established that, for the most popular imaging mode in optical microscopy, i.e. fluorescence; the diffraction barrier does no longer provide an unsurpassable limitation for resolution and localization accuracy. Furthermore, the terms "super resolution" and "optical nanoscopy", coined earlier, have been implemented in real far field optical microscopes, nowadays available for everyone to use without extreme complexity. Here, we will discuss targeted and stochastic readout methods using both single and multiphoton excitation, in terms of resolution and localization precision accuracy. Individual molecule localization (IML) implemented within selective plane illumination microscopy (SPIM) will be addressed towards 3D super resolution imaging in thick biological samples. STED two-photon excitation microscopy will be discussed reporting about the possibility of using a single wavelength (SW) both for two-photon excitation and STED depletion by implementing a SW-2PE-STED microscope. A further topic will be related to the coupling of STED and Atomic Force Microscopy. So far, a variety of architectures will be outlined in regard to specific applications demanding for nanoscale investigations.
- Harke B, Varghese Chacko J, Haschke H, Canale C, Diaspro A: A novel nanoscopic tool by combining AFM with STED microscopy. Optical Nanoscopy 1 (2012) 3.
- Bianchini P, Harke B, Galiani S, Vicidomini G, Diaspro A: Single wavelength 2PE-STED (SW-2PE-STED) super-resolution imaging. PNAS 109:17 (2012) 6390–93.
- Galiani S, Harke B, Vicidomini G, Lignani G, Benfenati F, Diaspro A, Bianchini P: Strategies to maximize the performance of a STED microscope. Opt Express 20:7 (2012) 7362–74.
- Bianchini P, Diaspro A: Fast scanning STED and two-photon fluorescence excitation microscopy with continuous wave beam. Journal of Microscopy 245:3 (2012) 225–228.
- Harke B, Bianchini P, Brandi F, Diaspro A: Photopolymerization Inhibition Dynamics for Sub-Diffraction Direct Laser Writing Lithography. ChemPhysChem 13:6 (2012) 1429–34.
- Mondal PP, Diaspro A: Simultaneous multilayer scanning and detection for multiphoton fluorescence microscopy. Nature Scientific Reports 1 (2011) 149/1–149/6.
- Cella Zanacchi F, Lavagnino Z, Perrone Donnorso M, Del Bue A, Furia L, Faretta M, Diaspro A: Live-cell 3D superresolution imaging in thick biological samples. Nature Methods 8:12 (2011) 1047–49.
- Cella Zanacchi F, Lavagnino Z, Pesce M, Difato F, Ronzitti E, Diaspro A: Two-photon fluorescence excitation within a light sheet based microscopy architecture. Proc SPIE 7903 (2011) 79032W.
Nano-organization of the AMPA receptors inside the synapse and physiological role
Eric Hosy, University of Bordeaux, CNRS, France
The majority of synapses in the central nervous system uses glutamate as a neurotransmitter, and the strength of synaptic transmission is proportional to the number of glutamate receptors (AMPA type) present under the synaptic glutamate release site. Many studies have reported modification of AMPA receptor quantity, organization or composition in response to various physiological stimuli which underlie synaptic maturation and plasticity, memory, disease, etc. However, available optical tools have not led to a precise description of the basic organization of receptors due to the limited pointing accuracy of the optical microscopy. The emergence of super-resolution techniques has broken this limitation barrier, allowing us to understand the organization of the AMPA receptors, and the variation of mobility as a function of its localization inside the synapse.
Here we used 3 different super-resolution techniques (STED, PALM and U-PAINT) to study extensively the organisation and the mobility of AMPAR inside the synapse and we discovered that AMPA receptor are not randomly distributed inside the synapse or even the PSD, but structured in nanodomains of about 80 nm. Such distribution allows maintaining a high fidelity of the synaptic response. In parallel, perturbation of one of the main scaffold protein of the PSD, PSD95, affect in the same range the dynamic organization of AMPAR and the synaptic currents.
Reduction of intensity demand for STED microscopy by using time-gated detection
Giuseppe Vicidomini, Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy
By causing adjacent features (closer than half of the wavelength of light, i.e. 200 to 300 nm) to fluoresce sequentially in time, stimulated emission depletion (STED) microscopy and other emerging super-resolution techniques have now substantially overcome the diffraction barrier. In STED microscopy, the sequential probing of fluorescent features is achieved by restricting the spatial extent of the volume from which fluorescence occurs. In practice, stimulated emission deprives the fluorescent molecules in the outer part of the excitation area of their ability to fluoresce. Since the stimulated emission transition has to compete with the spontaneous emission, relatively high intensities are needed for an effective fluorescence inhibition. Accordingly, STED microscopy has been most frequently implemented using pulsed STED beam (able to provide high peak intensity), which have rendered STED setup both costly and labor intensive. Moreover, high peak intensity can, in some cases, induce photodamage. Driven by this demand, it has been shown that STED microscopy can be implemented also with continuous wave (CW) beam. By using CW lasers, one can substantially simplify the implementation and lower both the cost and the peak intensity. Nevertheless, CWSTED was so far not reaching the same spatial resolution as pulsed-lasers STED configurations. Here we present two simple and complementary approaches to overcome this drawback of CW-STED microscopy. We simultaneously exploit the fact that the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event  and that the stimulated emission transition becomes more efficient by using STED beam with wavelength close to the emission peak of the fluorescent marker . Using a CW STED beam in conjunction with a pulsed excitation beam and time-gated detection allows us to improve the effective resolution of CW-STED and/or to reduce the STED intensity in the sample for a given resolution.
- Vicidomini G, Moneron G, Han KY, Westphal V, Ta H, Reuss M, Engelhardt J, Eggeling C, Hell SW: Sharper low-power STED nanoscopy by time gating. Nature Meth 8 (2011) 571–73.
- Vicidomini G, Moneron G, Eggeling C, Rittweger E, Hell SW: Sted with wavelengths closer to the emission maximum. Opt Expresss 20 (2012) 5225–36.
Deconvolution of STED images
Hans van der Voort, Scientific Volume Imaging BV, the Netherlands
Image restoration aims to make optimal use of the data provided by the microscope to recover the object being imaged, in other words, to "figure out what the microscope is actually trying to tell us". To achieve this, the distortions introduced by the imaging process need to be undone insofar possible. The most important one of these distortions is the resolution limiting effect of diffraction. In most microscope types diffraction results in a hard limit to which high spatial frequencies, corresponding with fine details in objects, are transmitted to the image. The success rate in restoring an object by deconvolution depends on the the 3D shape and extent of this limiting passband region, the need toand success rate in recovering lost frequencies beyond the passband region, and the object itself.
The STED microscope not only can extend the practical bandpass region of the confocal system on which it is based by a large factor, but, at least in theory, also dissolves the hard limit. The absence of a hard limit would seem to make deconvolution of STED data an easy job with potentially a huge gainin resolution over the raw data, gracefully limited by the noise level. However, the noise level in STED data tends to be high due to the limited number of photons available per pixel. Two more issues stand in the way of success: the Point Spread Function shape does not only depend on stable factors from the optics but also on more volatile factors like laser power and the specimen itself. Furthermore, thermal drift is often seen to cause the PSF to be deformed by sheering.
In this talk we will review the effects of diffraction in the confocal microscope, how the diffraction imposed resolution and bandwidth limits of various microscope types are related, and how the STED microscope breaks these limits.
Subsequently, we show how deconvolution methods can be constructed, and how deconvolution can circumvent diffraction imposed limits. Lastly, we show how the practical problems in STED imaging outlined above may be overcome, making it possible to obtain a significant resolution and contrast increase in a routine fashion with most STED data.