Abstract

The cyanobacterium Cyanothece sp. ATCC 51142 is the model photosynthetic bacterium being used for fundamental studies of solar energy capture process and conversion to the chemical energy [1, 2]. To facilitate processes such as photosynthesis, nitrogen fixation and carbon fixation during the circadian cycle, Cyanothece undergoes dramatic morphological changes [3]. As a part of Grand Challenge in Membrane Biology project, several research groups from different disciplines are working in an orchestrated manner to obtain data such as gene expression levels, proteomics and metabolomics during the circadian cycle. We are creating a whole cell 3D model to provide basis for modeling dynamic cellular processes and correlate Cyanothece ultrastructural changes with these molecular networks datasets. We are using TEM tomography as a tool for constructing this 3D model. The Cyanothece cell is of a spherical shape about 3 um in diameter, and thus consecutive serial sectioning is necessary for obtaining the whole cell model. Individual TEM tomograms from consecutive plastic sections are then aligned into one whole cell volume. Feature segmentation and cell components rendering will provide a comprehensive model of Cyanothece's internal architecture. The main point of interest is the organization of the intricate network of thylakoid membranes, as well as spatial distribution of an assortment of storage granules and other cell components [4].Cyanothece cells grown in alternating 12 hour light and dark intervals were high pressure frozen, freeze substituted and embedded in Epon. Ribbons of serial 200 nm thick sections were mounted on a copper slotted grid, and inspected in Tecnai T-12 (LaB6) with tomography stage. 2x2K CCD camera (Gatan) was used for imaging and analyses. Tilt series were acquired typically from +/-70 degrees, single tilt, taking advantage of a large opening in the slotted grid that didn't interfere with the tilting window if sections ribbon was positioned in the central portion of a grid parallel with the longer oval grid opening dimension. We are aware of the fact that the higher accelerating voltage TEM would enable the 3D reconstruction of a whole bacterial cell in fewer steps. However, with instrumentation present in our facility, and the further focus on much smaller cellular components such as phycobilisomes and ribosomal trafficking along the thylakoid membranes, we have decided to utilize our TEM for this project, regardless of the possible disadvantage of more thin sections required for the whole cell reconstruction. Our findings show that the 120kV instrument provided excellent contrast and resolution for this purpose. Regardless of the method of acquirement of the individual tomography volumes, the most time-consuming and critical component of the visualization process is the features segmentation. To aid the manual segmentation process, a series of image analysis and 1338 CD