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Super-resolution optical fluctuation imaging (SOFI) is a super-resolution microscopy technique that overcomes the diffraction limit by analyzing intensity fluctuations of statistically independent emitters in a time series of images. The final images are background-free and show confocality and enhanced spatial resolution (super-resolution). Fluorophore photobleaching, however, is a key limitation for recording long time series of images that will allow for the calculation of higher order SOFI results with correspondingly increased resolution. Here, we demonstrate that photobleaching can be circumvented by using fluorophore labels that reversibly and transiently bind to a target, and which are being replenished from a buffer which serves as a reservoir. Using fluorophore-labeled short DNA oligonucleotides, we labeled cellular structures with target-specific antibodies that contain complementary DNA sequences and record the fluctuation events caused by transient emitter binding. We show that this concept bypasses extensive photobleaching and facilitates two-color imaging of cellular structures with SOFI.

Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal-to-noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed by using exchangeable labels, which transiently bind to and dissociate from a target, thereby replenishing the destroyed labels with intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore-labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein-specific antibodies. The constant exchange of fluorophore labels in DNA-based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two-color STED imaging of whole cells.

We demonstrate super-resolution imaging of proteins and nucleic acids that were densely labelled with fluorophores using the concept of “click chemistry”., We demonstrate high-density labelling of cellular DNA and RNA using click chemistry and perform confocal and super-resolution microscopy. We visualize the crescent and ring-like structure of densely packed RNA in nucleoli. We further demonstrate click chemistry with unnatural amino acids for super-resolution imaging of outer-membrane proteins of E. coli.

From the Summar of the Report Production At worldwide level in year 2003 66% of the world’s organic land (total 24 mio ha) are concentrated in two continents: Australia and Latin America. In these regions extensive grazing land is widespread beside the whole game of plant production, from cereals to coffee, tea and other tropical products. In Northern America the organic farmland achieves 1,5 mio ha. Europe has 23 % of the worldwide organic land (5,5 mio ha), these 5,5 mio ha correspond to almost 2% of agricultural land managed organically. Liechtenstein is with 26 % of agricultural area managed organically the leader followed by Aus-tria with 11% and Switzerland with 10%. In most countries the organically managed surface is still increasing. In the enlarged EU the area raised 4% from year 2002 to 2003, in the new countries of the EU (Poland, Estonia, Lithuania and Hungary) the growing rates of organic farm land is over 10%. Small declines have been observed in Denmark, The Netherlands, Italy and United Kingdom. In the European Union and Switzerland the agrarian policy supports organic sec-tor with different measurers like direct payments for organically managed area, contribution for conversion, payments for environmental services and animal wel-fare, training and capacity building, research programs etc. Ukraine was the granary of the former Soviet Union. Large surfaces of fertile black soils are favourable for organic farming. In Ukraine 230’000 ha are certified organic or in conversion in year 2003. This represents 0.58% of land area under organic management. 69 production units with an average size about 3’500 ha are certified. Wheat, barley, sunflower and corn are the most important crops on these farms. The organic production increased in the last years with the expecta-tion to get access to export markets. In Ukraine certified organic fruit and vegetable producers are missing. There is also nearly no animal husbandry farm certified. The potential for conversion of plant production units is high, because many farmers use few external inputs. For smaller farms, without access to export markets, the certification costs of international certifiers are unattainable. Until now the agrarian policy doesn’t support especially organic farming. The ministry of agriculture and the commission of the parliament for agriculture are in contact with the new organic farming association Biolan Ukraine and other stake-holders for the elaboration of a law for organic production. There is advisory and training capacity for organic farming but this is still insufficient for the whole country. Market overview Europe and Northern America are responsible for 97% of organic sales to con-sumers. There is a global harmonisation of market trends and buying behaviours. The organic consumer lives in urban centres, has a higher education which is transformed in higher income, is young and has children or is older with a healthy lifestyle. Important issues for this consumer are food and health (free of dangersafety, label, certification) and food and emotions (animal welfare, regional product). Important triggers for buying organic are children, allergies and healthy life-style. The barriers for not buying organic are the price, the availability and in general less importance given to nutrition. Between the countries there are significant differences in the importance of the sales channels from direct marketing included weekly markets and box schemes, specialised organic food shops to general food shops (retailer shops, supermarkets, and discounter). General food shops are the most important sales channels over all and especially in countries with mature food markets like Austria, Den-mark, France, Switzerland and United Kingdom. Germany with a well developed organic food market is an exception with only 35 % of sales in general food shops. The organic market is in a growing phase in Northern America (15%-30%), Italy, The Netherlands, Norway, Sweden, Portugal and Finland. The rest of the Euro-pean countries, the Ukrainian neighbours like Russia and the Baltic States, Asian countries like Korea and Chine and Middle East countries like Saudi Arabia are in the phase of emerging market. The most important markets (in value and in decreasing order) are: USA, Ger-many, United Kingdom, France, Italy, Canada and Switzerland. In Switzerland the average consumer spends 104 Euro on organic products per annum, this is the highest amount. The price premiums for organic food paid to the farmers in the EU countries in year 2000 vary tremendous between the countries and the products. For cereals the average was 102% and the highest average price premium for plant product was reported for potatoes with 257%. The price premiums for animal products paid in EU are in average lower (milk 22%, beef 34% pork 68%) than for plant products with exception of poultry with 182% and eggs with 167%. In Europe there are supply and demand imbalances: oversupply in milk and beef and supply gaps in cereals. European organic fruit, vegetables and cereals can normally be sold as organic within Europe. Tropical, off season and exotic (eth-nic) products are imported to Europe and Northern America. In Europe the self sufficiency degree shows big variations from country to country and product to product. In cereals for example in year 2001 Belgium has 2% self sufficiency and Spain 316%, France an exporter of conventional cereals reports self sufficiency degree for organic of 35%. Even more important then the self suf-ficiency degrees are supply gaps (national production and imports are not suffi-cient). In the OMIaRD market research there were expected the following supply deficit for year 2003/04: For wheat and barley in Germany and Slovenia, for rye in Slovenia and Finland, for oilseed in Germany, Finland and Sweden, for legumi-nous fodder crops in Austria, Germany, Italy, The Netherlands and United King-dom. Access to the EU and Swiss market is possible when the products are certified according to EU-standard (EU-regulations 2092/91 and 1804/99) or Swiss or-ganic ordinance. Depending on the market, other, mainly private, standards need to be fulfilled In Ukraine the national market for organic products is in the initial phase with some imported products like baby food, tea or coffee. The potential organic con-sumers are urban, younger professional women and young families with small children, from the new middle to upper class. At the moment they buy so called environmental clean products, with 20% to 100% price premium. These products are not certified; they are supposed to come from regions without relevant human made pollution and free of radioactivity. The Ukrainian consumers are aware of important organic issues like health and dangers residues. Up to now “organic” is not protected by law. Experts estimate that 5% to 10% of the Ukrainian products certified organic (mainly cereals and oilseeds) are sold as organic and exported to Western Europe.

Die Studie zur Ermittlung von derzeitigen und absehbaren Vermarktungsproblemen im Markt für ökologisch erzeugtes Rindfleisch wurde mit der Forschungsmethode einer „mehrstufigen Befragung nach dem Delphi-Prinzip“ bearbeitet. Den Befragungen war eine umfassende Analyse der Veröffentlichungen über den Markt für ökologisch erzeugtes Rindfleisch vorausgegangen. Die Ergebnisse zeigen, dass die Branche sich in einem strukturellen Engpass befindet. Das individualistische Agieren zahlreicher kleiner Akteure stößt im größer werdenden Markt und angesichts zunehmender Komplexität an Grenzen. Es gibt Ansätze von verstärkter Kooperation, die auch mit Erfolg am Markt belohnt werden. Die Darstellung der Probleme im Ergebnisteil hat gezeigt, dass viele Probleme wie uneinheitliche Verbraucheransprache und fehlende Qualifikationen durch die Struktur des Ökomarktes allgemein entstanden sind. Andere Probleme wie Marktrisiken und Richtlinien liegen außerhalb des Einflussbereichs der Marktteilnehmer. Als ein zentrales Ergebnis, das sich wie ein roter Faden durch die Studie zieht, ist der Mangel an Kommunikation innerhalb der Branche und damit verbunden eine genereller Informationsmangel. Die vorgeschlagene Lösung, die als Schlüssel für einige angesprochene Schwierigkeiten gelten kann, ist der horizontale und vertikale Zusammenschluss von Partnern mit ähnlichen Interessen. Ein weiteres zentrales Problem könnte mit dem Begriff „Rechtsunsicherheit“ umrissen werden. Die Marktteilnehmer fühlen sich angesichts der Mängel der so genannten „EU-Öko-Verordnung“ (abweichende Produktions- und Kontrollstandards innerhalb benachbarter Märkte) stark verunsichert. Die Schwächen des bestehenden Kontrollsystems, das als zu schwerfällig, als zu ineffizient, als zu uneinheitlich innerhalb Europas aber auch innerhalb Deutschlands betrachtet wird, verstärken diese Unsicherheit.

Die Studie wurde mit der Forschungsmethode einer „mehrstufigen Befragung nach dem Delphi-Prinzip“ bearbeitet. Den Befragungen vorausgegangen war eine umfassende Analyse der Veröffentlichungen über den Markt für ökologisches Schweinefleisch. Die Resultate aus den Befragungen haben eine Problem- und Schwachstellenanalyse des Marktes ergeben. Für zehn Problemkreise wurden die aktuelle Bedeutung, Problempotentiale und Wirkungen und auch Lösungsansätze beschrieben. Die zehn Problemkreise sind: - Qualität - Rückverfolgbarkeit - Kommunikation - Verbraucheransprache - Marktrisiko und Kapitalisierung - Züchtung und Ferkelbeschaffung - Teilstückproblematik - Wirtschaftlichkeit und Markt - Richtlinien - Zusatzstoffe Die Darstellung der Probleme im Ergebnisteil hat gezeigt, dass viele Probleme wie uneinheitliche Verbraucheransprache und fehlende Qualifikationen durch die Struktur des Ökomarktes allgemein entstanden sind. Andere Probleme wie Marktrisiken und Richtlinien liegen außerhalb des Einflussbereichs der Marktteilnehmer. Auffällig war eine große Unsicherheit bei der Einschätzung von Marktvolumen und -entwicklung, von Wettbewerbern und der eigenen Positionierung. Mit einer Untersuchung des Marktes für Ökoschweinefleisch in Dänemark werden die Unterschiede zum deutschen Markt deutlich. Die dänische Vorgehensweise wie Überwachung der Futtermittel, des Gesundheitsstatus der Tiere und der verabreichtren Arzneimittel durch den Staat sowie ein landesweit einheitliches Qualitäts- und Gesundheitsmanagement sollte als Vorbild für Deutschland fungieren. Als ein zentrales Ergebnis, das sich wie ein roter Faden durch die Studie zieht, ist der Mangel an Kommunikation innerhalb der Branche und damit verbunden eine genereller Informationsmangel. Die vorgeschlagene Lösung, die als Schlüssel für einige angesprochene Schwierigkeiten gelten kann, ist der horizontale und vertikale Zusammenschluss von Partnern mit ähnlichen Interessen. Ein weiteres zentrales Problem könnte mit dem Begriff „Rechtsunsicherheit“ umrissen werden. Die Marktteilnehmer fühlen sich angesichts der Mängel der so genannten „EU-Öko-Verordnung“ (abweichende Produktions- und Kontrollstandards innerhalb benachbarter Märkte) stark verunsichert. Die Schwächen des bestehenden Kontrollsystems, das als zu schwerfällig, als zu ineffizient, als zu uneinheitlich innerhalb Europas aber auch innerhalb Deutschlands betrachtet wird, verstärken diese Unsicherheit.