VISUALIZE MARINE DIATOM ULTRASTRUCTURE OR EXTRACELLULAR MATRIX BY CRYO STEM

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We are currently funded by the Office of Naval Research to investigate the production of bioadhesives by algae. Our project involves the biofouling diatom Achnanthes longipes. On of the objectives in this research is to determine the structure and mechanism of biogenesis of the specific components of diatom extracellular adhesives. In order to accomplish this ob ective we have utilized several techniques including DIC video time-lapse and fluorescence microscopy coupled with traditional electron microscopical techniques. We have also developed/implemented specific molecular probes directed against the adhesives including monoclonal antibodies, pectins and enzymes. Although we are interested in potential applications to our research of other techniques available at the HAR, we have need for two methods now. The adhesives of Achnanthes are very hydrophilic and undergo significant morphological change when exposed to standard electron microscopical dehydration procedures for TEM and SEM. In order to observe the bulk adhesive without these dehydration artifacts we require cryo-SEM technology not available at NITU. We also have been largely unsuccessful at visualizing the site of adhesive synthesis with the goal of further characterizing the inh-acellularly using standard TEM fixation techniques. We wish to attempt high-pressure freezing of Achnanthes actively involved in adhesive synthesis with the goal of further characterizing the intracellular structures involved. We plan to bring live tissues to the IMR with the intent to do the cryo-SEM observations on site. We would also like to utilize IMR facilities to do high pressure freezing and the tissue would be further processed for TEM at MTU.