KIT – University of the State of Baden-Wuerttemberg and National Research Center of the Helmholtz Association Investigation of motile marine organisms with Digital In-line Holographic Microscopy in situ and under lab conditions Svenja Stuppy 1,3* , Matthias Heydt 1 ,Geoffrey W. Swain 4 , J.A. Callow 5 , M.E. Callow 5 , Michael Grunze 1,2 , Axel Rosenhahn 1,3 1 Applied Physical Chemistry, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany 2 Institute for Toxicology and Genetics, ITG, Karlsruhe Institute of Technology, PO Box 3640, 76021 Karlsruhe, Germany 3 Institute for Functional Interfaces, IFG, Karlsruhe Institute of Technology, PO Box 3640, 76021 Karlsruhe, Germany 4 Ocean Engineering and Oceanography, Florida Institute of Technology, 150 West University Boulevard, Melbourne Florida 32901, USA 5 School of Bioscience, University of Birmingham, Birmingham B15 2TT, UK Acknowledgment • Thanks to the group of Prof. Swain Florida Institute of Technology • The work was funded by Office of Naval Research (Grant number N00014-08-1-1116) and DFG 252412-2 Introduction Biofouling caused by different micro- and macro-organisms such as algae, diatoms or invertebrates is a major problem in marine industries [1]. The investigation of the settlement behavior of these organisms on different surfaces or coatings allows to reveal selection strategies. In previous work swimming and settlement behavior of Ulva linza zoospores under lab conditions was observed in the vicinity of different chemistries with a digital in-line holographic Microscope [2]. For the investigation of marine organisms in their native environment experiments on a test facility situated at the Indian River Lagoon in Melbourne Beach Florida were performed. Field experiments Holography Reconstruction Resolution L 2 D NA = Hologram 3D-Projection Kirschhoff-Helmholtz Transformation • Transportable holographic Setup in in-line geometry, first proposed by Gabor in 1948 [3] • Experiments performed in a mobile laboratory (working in a Van) • independent from electrical power supply; working with car batteries • Seawater taken out of seawater tank and filtered with 30μm netfilters •Record of Hologram series with up to 18000 frames (10 frames per second ) Detector (CMOS Chip) DPSS Laser 532 nm Apertures Beamexpander 20x Objectiv Sample translationstage 500nm Pinhole Mirrors Source Hologram Twin image Self interference 2 sc sc in sc in 2 in ) ( ) ( ) ( ) ( ) ( ) ( ) ( r r r r r r r I        Ψ + Ψ Ψ + Ψ Ψ + Ψ = ∗ ∗ ξ d ξ r ξ ik exp ) ξ I( ) r K( s 2 ∫         =     NA 0,61λ δ lateral = 2 depth NA λ δ = Tracking Marine Biofoulers 71.75 284.8 Ulva Linza spore on AWG Orientation pattern Setup The motile zoospores of the green algae Ulva linza are established model organisms to investigate exploration behaviour and settlement dynamics related to different chemical cues. Settlement is a critical stage in Ulva life cycle. The selection of a suitable surface is important for their survival and reproduction. However Ulva linza has to be collected from a seashore prior to experiments. The zoospores can be released and measured in the laboratory at University of Birmingham. The step from a model organism to a conglomeration of marine biofoulers under native conditions is a big challenge but necessary to correlate between lab and field. Comparison Lab versus Field Lab [4] Field Mean velocity of tracked organisms Round organism Orientation pattern Field Lab Lab Marine organisms after 3 h in observation chamber Ulva spores after 35 s in observation chamber Ulva spores after 11:57 min in observation chamber Literature: [1] Rosenhahn, A., Ederth, T. & Pettit, M. E. Advanced Nanostructures for the control of biofouling: The FP6 EU Integrated Project AMBIO. Biointerphases 3, IR1-IR5 (2008) [2] Heydt, M. et al. Digital in-line holography as a three-dimensional tool to study motile marine organisms during their exploration of surfaces. Journal of Adhesion 83,417-430 (2007) [3] Gabor, D. (1948). "A new microscopic principle." Nature 161: 777-778 [4] Matthias Heydt How do spores select where to settle? Springer Theses ISBN 978-3-642-17216-8 Outlook Observation of active swimming organisms with elongated shape Velocity coded 3D-trajectories Velocity distributions of Ulva linza on AWG and spherical undefined organisms measured in the field •Velocity of Ulva linza spores under lab conditions shows a split in a slow and a fast swimming fraction after longer incubation time in the observation chamber •Organisms in field experiment show a broader velocity distribution without spliting even after a very long incubation time •Velocity distribution of organisms with elongated shape splits in two fractions fast swimmers (mean velocity: 284.8 μm/s) and slow swimmers (mean velocity:71.75 μm/s) •Elongated organisms swim along thier longitudinal axis and have a round shape relating to their transversal axis 3D trajectory with shape of organisms observed in xy-projections •Reproduction and further investigations of settlement behaviour of Ulva linza spores on surfaces with different surface properties •Differentiation and possibly identification of marine species from field experiments on the basis of their shape and velocity •Further correlation of field work with lab experiments •Correlation of holographic data with other data obtained in the field (settlement kinetics, Microfluidics, Conditioning film formation, ect.) Organism with elongated shape Spherical organism xy-projection *email: svenja.stuppy@pci.uni-heidelberg.de