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Sunday Afternoon, October 28, 2012 Sunday Afternoon, October 28, 2012 Biomaterials Plenary Session on-off-switching at low intensities I, by resorting to molecular switching mechanisms that entail low switching thresholds Is. An Is lower by many Room: 23 - Session BP+AS-SuA orders of magnitude is provided by reversibly switching the fluorophore to a long-lived dark (triplet) state2 or between a long-lived ‘fluorescence Biomaterials Plenary - Bioimaging: In Vacuo, In Vitro, activated’ and ‘deactivated’ state.2,5 These alternative switching mechanisms entail an Is that is several orders of magnitude lower than in In Vivo STED. In imaging applications, STED/RESOLFT enables fast recordings Moderator: M.R. Alexander, University of Nottingham, and the application to living cells, tissues, and even living animals.6,7 UK Starting from the basic principles of nanoscopy we will discuss recent developments8,9 with particular attention to RESOLFT and the recent 7 4:00pm BP+AS-SuA1 NanoBio Imaging for New Biomedical nanoscale imaging of the brain of living mice by STED. Applications, D.W. Moon, Korea Research Institute of Standards and 1 Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by Science INVITED stimulated-emission - stimulated-emission-depletion fluorescence Surface and interface analysis techniques have been mainly developed to microscopy, 780-782, doi:10.1364/OL.19.000780 (1994). meet the demands on atomic scale characterization from semiconductor 2 Hell, S. W. Toward fluorescence nanoscopy, 1347-1355 (2003). industries. KRISS has been trying to meet the surface and interface analysis 3 Hell, S. W., Jakobs, S. & Kastrup, L. Imaging and writing at the challenges from semiconductor industries and furthermore to extend the nanoscale with focused visible light through saturable optical transitions, application scope to biomedical areas. In this presentation, I’d like to report 859-860 (2003). our recent activities of nanobio imaging for new biomedical applications such as 1) Coherent Anti-Stokes Raman Scattering (CARS) for 4 Hell, S. W. Far-Field Optical Nanoscopy, 1153-1158 (2007). atherosclerotic plaque imaging 2) Time-of-flight secondary ion mass 5 Hofmann, M., Eggeling, C., Jakobs, S. & Hell, S. W. Breaking the spectrometry (TOF-SIMS) for mass imaging of collagen fibrils, diffraction barrier in fluorescence microscopy at low light intensities by atherosclerotic plaques, and cancer tissues and 3) Surface Plasmon using reversibly photoswitchable proteins, 17565-17569 (2005). Resonance Imaging Ellipsometry for cell adhesion, migration, and 6 Rankin, B. R. Nanoscopy in a Living Multicellular Organism Expressing infiltration dynamics for HUVEC, CASMC, and T cells 4) TOF-medium GFP, L63 - L65 (2011). energy ion scattering spectroscopy (TOF-MEIS) for nanothin films and nanoparticles such as CdSe/ZnS quantum dots and calcium hydroxyapatite 7 Berning, S., Willig, K. I., Steffens, H., Dibaj, P. & Hell, S. W. Nanoscopy nano-size biominerals. Future challenges of nanobio imaging for biomedical in a Living Mouse Brain, 551 (2012). applications will be discussed. 8 Grotjohann, T. Diffraction-unlimited all-optical imaging and writing with a photochromic GFP, 204-208 (2011). 4:40pm BP+AS-SuA3 3-D View into Cells by X-ray Nano- 9 Brakemann, T. A reversibly photoswitchable GFP-like protein with Tomography, G. Schneider, P. Guttmann, S. Werner, K. Henzler, S. fluorescence excitation decoupled from switching, 942-947 (2011). Rehbein, Helmholtz-Zentrum Berlin für Materialien und Energie GmbH, Germany INVITED X-ray imaging offers a new 3-D view into cells. With its ability to penetrate whole hydrated cells it is ideally suited for pairing fluorescence light microscopy and nanoscale X-ray tomography. The HZB TXM at the undulator U41 provides a spectral resolution of 10.000 and a spatial resolution of 11 nm. For high resolution tomography, we adopted a tilt stage originally developed for electron tomography. The stage is able to tilt samples up to ± 80°. Such a large tilt of flat sample holders is impossible with TXM at bending magnet sources because they require a monochromator pinhole to be positioned close to the specimen. In our TXM, the holder geometry is no longer restricted to glass tubes.Conventional fluorescence images are diffraction-limited to ~200 nm, whereas current TXM achieve a ten-fold improvement in resolution. Since fluorescence and X-ray microscopy permit analysis of whole cells, it is possible to investigate the same cell in both microscopes by correlative microscopy. These correlative studies are ideally suited to X-ray microscopy because of its ability to image cells in 3D. In the talk, we present the cryo TXM and selected applications. In particular, we will show the internal structures of mammalian cells, i.e. plasma membrane,nuclear membrane, nuclear pores, nucleoli, endoplasmic reticulum, vesicles, lysosomes and mitochondria. It is now also possible to resolve internal organellar structures, such as mitochondrial cristae, the double nuclear membrane and lysosomal inclusions. In addition, we discuss ways towards 10 nm 3D imaging of cells. Keywords: X-ray microscopy, tomography, cell organelles, correlative microscopy References 1. S. Rehbein, S. Heim, P. Guttmann, S. Werner, G. Schneider, Phys. Rev. Lett. 103, (2009) 110801 2. G. Schneider, P. Guttmann, S. Heim, S. Rehbein, F. Mueller, K. Nagashima, J.B. Heymann, W.G. Müller, J.G. McNally, Nature Methods 7 (2010), 985-987 3. P. Guttmann, C. Bittencourt, S. Rehbein, P. Umek, X. Ke, G. Van Tendeloo, C. P. Ewels and G. Schneider, Nature Photonics 6 (2012), 25-29 4. G. Schneider, P. Guttmann, S. Rehbein, S. Werner, R. Follath, J. Struct. Biol. 177 (2012), 212-223 5:20pm BP+AS-SuA5 Nanoscopy with Focused Light, S.W. Hell, Max- Planck-Institut für Biophysikalische Chemie, Germany INVITED In STED microscopy1, 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. In RESOLFT microscopy,2,3 the principles of STED have been expanded to fluorescence 1 Sunday Afternoon, October 28, 2012 Monday Morning, October 29, 2012 Applied Surface Science nanomorphology can be extracted from the photoelectron angular/energy distribution is investigated, in particular the question is addressed whether Room: 20 - Session AS-MoM the average size of nanostructures on a surface is accessible by means of analysis of angular/energy photoelectron spectra. This is done for standard Quantitative Surface Chemical Analysis, Technique AlKα and Mg Kα laboratory sources, but the possibility to gain additional information by increasing the photon energy to the hard x-ray regime is also Development, and Data Interpretation - Part 1 examined. Moderator: J.A. Ohlhausen, Sandia National Laboratories, 1. http://www.nist.gov/srd/nist100.cfm. S. Suzer, Bilkent University, Turkey 2. W. Smekal, W. S. M. Werner, and C. J. Powell, Surf. Interface Anal. 37, 1059 (2005). 8:20am AS-MoM1 2012 AVS Albert Nerken Award Lecture: Characterization of Thin-Film Nano-Structures by XPS, S. Tougaard*, 9:20am AS-MoM4 XPS Characterisation of InP Features Etched in University of Southern Denmark INVITED Cl2-Ar and Cl2-H2 Inductively Coupled Plasmas, C. Cardinaud, CNRS, This is a brief summary of the work that was involved in the development France, R. Chanson, CNRS-IMN, France, S. Bouchoule, CNRS-LPN, of the technique for quantitative XPS from analysis of the background of France, A. Rhallabi, M.-C. Fernandez, Université de Nantes, France inelastically scattered electrons. About 30 years ago it became evident that High-aspect-ratio etching of InP-based heterostructures is a critical building these electrons must carry valuable information about the depth where the block for photonic device fabrication. This study is focused on the chemical XPS electrons are excited. Theoretical modeling started and algorithms characterisation of the bottom and sidewall surfaces of InP ridge patterns were developed. It was necessary to have an accurate description of the etched with Cl2-Ar and Cl2-H2 plasmas using a SiNx mask. Each sample electron energy loss processes which at that time was not available. contains five arrays combining various ridge widths (1.5 to 4µm) and space Theoretical calculations of inelastic cross sections based on a dielectric widths (5 to 16µm) plus four InP and mask open areas. Experiments are response description were done and a new experimental method to carried out in a Kratos-Axis-Ultra. The direction of analysis is vertical, i.e. determine this from analysis of reflected electron energy loss spectra normal to the sample surface, while the x-rays strike the surface with an (REELS) was also developed. angle of incidence α = 60°. The sample can be rotated in azimuth to align To make these procedures for quantitative XPS analysis work in practice it precisely the arrays, either parallel to or perpendicular to the plane defined is however not possible to use calculations valid only for specific sample by the x-source and the analyser. The first arrangement enables the analysis compositions. Therefore an effort was made early on to find cross sections of the bottom (as well as the top of the ridge), whereas the second which can be used as an approximation for wide classes of materials and arrangement allows the analysis of the sidewall after tilting the sample. In compositions. This resulted in the Universal cross sections which are now this latter case two configurations are used. Taking advantage of the widely used and without which practical use of the formalism would have absorption of the x-rays by the InP ridges (1.8µm of InP absorbs 99% of been very limited. The resulting XPS analysis technique was summarized in AlKα under 60° of angle of incidence), the first configuration consists in [1]. tilting the sample opposite to the x-ray source until the bottom is totally In the following years, much effort was then centered on applications to screened. Simultaneously this brings the sidewall that is irradiated in the increasingly finer details of the morphology of nanostructures.
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