In cyanobacteria, activation of the Orange Carotenoid Protein (OCP) by intense blue-green light triggers photoprotective thermal dissipation of excess absorbed energy leading to a decrease (quenching) of fluorescence of the light harvesting phycobilisomes and, concomitantly, of the energy arriving to the reaction centers. Using spectrally resolved picosecond fluorescence, we have studied cells of wild-type Synechocystis sp. PCC 6803 and of mutants without and with extra OCP (ΔOCP and OverOCP) both in the unquenched and quenched state. With the use of target analysis, we managed to spectrally resolve seven different pigment pools in the phycobilisomes and photosystems I and II, and to determine the rates of excitation energy transfer between them. In addition, the fraction of quenched phycobilisomes and the rates of charge separation and quenching were resolved. Under our illumination conditions, ∼72% of the phycobilisomes in OverOCP appeared to be substantially quenched. For wild-type cells, this number was only ∼29%. It is revealed that upon OCP activation, a bilin chromophore in the core of the phycobilisome, here called APC(Q)(660), with fluorescence maximum at 660 nm becomes an effective quencher that prevents more than 80% of the excitations in the phycobilisome to reach Photosystems I and II. The quenching rate of its excited state is extremely fast, that is, at least (∼240 ± 60 fs)(-1). It is concluded that the quenching is most likely caused by charge transfer between APC(Q)(660) and the OCP carotenoid hECN in its activated form.

Microfabrication has its foundations in microelectronics, where photolithography, alongside other techniques, is used to make microfabricated circuits. For use in biology, Xia and Whitesides have developed soft lithography methods to fabricate micropatterned stamps from elastomeric materials [1] which were then employed to print biofunctional molecules on cell culture dishes and glass, [2] mold a second material (replica molding) [3] and make miniaturized flow cells, i.e., microfluidic devices. [4,5] Owing to these developments, the fabrication of micro-and nanostructures for use in biology has become a large area of research spanning a wide range of applications, including biochemical assays and studies of cell adhesion and spreading. [6] Regarding the latter, surface chemistry, topography, and elasticity are known to affect and direct cell adhesion, migration, proliferation, and even differentiation. [7][8][9][10][11][12][13][14] An understanding of these factors and how they induce intracellular processes is a fundamental requirement when designing biomaterials for biomedical applications.In our studies of cellular behavior on micro-and nanopatterned hydrogels we have discovered that a surface topographic pattern induces adhesion and spreading of fibroblasts on intrinsically anti-adhesive poly(ethylene glycol) (PEG) gels. [15,16] In addition, smooth but elastically micropatterned PEG-based hydrogels enabled fibroblast adhe-We have employed our recently developed method Fill-Molding In Capillaries (FIMIC) to fabricate elastically micropatterned substrates, using two poly(ethylene glycol) (PEG)-based polymers with different elastic properties and swelling behavior. We have evaluated the FIMIC process and the quality of the eventual substrates (the ''FIMICs'') by atomic force microscopy (AFM); imaging the surface topography and quantifying the local surface elasticity. Topographical imaging reveals that the surface of the FIMICs is never perfectly smooth; a slight topographic difference of 30 nm up to several hundreds of nm is observed, with the filler material always being depressed with respect to the mold. Moreover, when the FIMICs are immersed in water (or cell culture medium), the topographical landscape changes due to differential swelling of the two constituents of the FIMICs. We have used this differential swelling to our advantage in order to diminish the topography differences present on the sample surface by employing a filler that swells more than the mold. Finally, cell culture experiments with fibroblasts underlines the topographical influence on cell adhesion on the more or less anti-adhesive PEG-based materials.B56

No abstract