"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.
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 (