Single computed x-y slice through a tomographic volume of the chloroplast of a blue green algae exposed to low light conditions. The quality of the reconstruction is not very good due to alignment difficulties.
Full resolution image description
Zip file containing volume file in Analyze 7.5 format. Gtomo outputs the volume as a series of slices in the x-z plane. The volume has not been resliced in the xy plane. Curator's note: the quality of the reconstruction is not very good; it looks like there were alignment difficulties.
550, 290, 300
Animation through the slices in the xy plane of a tomographic volume of blue green algae exposed to low light conditions.
Zero degree tilt image of a 0.75 um thick section of blue green algae imaged using intermediate voltage electron microscopy. Contrast is reveresed so that electron dense structures appear bright. The small bright particles are colloidal gold particles that were placed on the section surface to serve as fiducial cues. Contrast was enhanced from original data for display purposes.
Full resolution image description
Zip file containing the unaligned, full size digitized images (*.f), the aligned full size images (*.s), the aligned cropped images used for reconstruction (*.crop) along with the normalized (*.norm) and radius weighted (*.w) images used for reconstruction. Also included are the fiducial mark file (*.fido) and the list of angles used for reconstruction (*.ang). This file is ~ 300 Mb in size.
Chloroplast Ultrastructure of Phaeocystis antarctica in High and Low Light Conditions
The three-dimensional morphological rearrangements for two conditions that mimic light conditions for the Antarctic summer and winter were studied in Phaeocystis antarctica Karsten
National Aeronautics and Space Administration
Low light condition
To examine the architecture of thylakoid membranes in algae grown under low light conditions
Microscopy product ID
JEOL 4000EX IVEM
continuous blue light 14 quanta m-2 s-1.
Electron microscopy product
Need to check the magnification
Culture conditions. Cultures of colonial P. antarctica (CCMP 1374) were grown semi-continuously for 5-8 generations in f/2 medium (Guillard and Ryther 1962) under continuous blue light at 4C at irradiances of 14 and 259 mol quanta m-2 s-1.Specific growth rate. Specific growth rate was estimated by a linear regression of loge transformed daily determinations of in vivo fluorescence intensity (n=2) measured with a Turner Model 10 fluorometer. Sample preparation for electron microscopy. P. antarctica colonies were fixed on ice with a 2% glutaraldehyde and 1.3% osmium tetroxide solution for 30 minutes and rinsed in distilled water. Cells were dehydrated through a series of ethanol: water washes (25:75, 50:50, 75:25, 95:5), three 100% ethanol washes and finally through three washes of 100% acetone. Cells were pelleted and fixed in an Epon resin. The fixation process lends itself to a breakup of the colonial matrix and we were able to examine P. antarctica individual colonial cells using electron tomography. Embedded samples were cut on a Reichert-Jung Ultracut E microtome, transferred to 50/50 mesh copper clam grids, and stained with uranyl acetate and lead citrate. After staining, 20 nm colloidal gold particles (Sigma-Aldrich Chemicals, St. Louis, MO) were added to both sides of the grid to serve as fiducial markers for aligning tilted images. Individual colonial cells were observed at low magnification at 80kV on a JEOL 100CX to determine specimen quality and to select suitable samples. Intermediate voltage electron microscopy. Sections of 0.25 (high light condition) and 0.75 m (low light condition) in thickness were cut, post-stained with uranyl acetate and lead citrate and examined at 400 kV on a JEOL 4000 intermediate voltage electron microscope. Tilt series consisting of 61 images (-60 to 60 at 2 tilt increments) were collected at either 12-15,000 magnification (low light condition) or 20-30,000 magnification (high light condition). Images were collected on film (Kodak 4489 electron image film) or on a Slow-Scan Cooled CCD camera (Fan et al. 2000). Sections were pre-irradiated before each tilt series in order to limit anisotropic specimen thinning during specimen examination (Luther 1992). The illumination was held constant using parallel electron beam conditions and the image was maximized for each exposure. A computer-controlled goniometer was used to accurately tilt the specimen. For tilt series acquired on film, digitization was accomplished using a Photometrics 1024 x 1024 Cooled CCD camera containing a 19-m2 pixel with sampling sizes of ~50-85 m pixel-1.Single-axis tilt series tomographic reconstruction methodology. Tilted images were aligned with each other by use of a set of common fiducial marks consisting of 20 nm colloidal gold beads. Reconstruction methods follow that those of Perkins et al. (1997). The common fiducial marks on each image of the tilt series were aligned using the program XFIDO. Alignment of the tilt series was initially calculated using a least-squares algorithm through the z-direction of the tilt series using the program SAXALIGN. After initial alignment, volumes were computed using either a standard r-weighted simple back projection algorithm or a Globus enabled parallelized version of this algorithm that considerably speeded up these computations (Smallen et al. 2000).The 3D reconstruction is viewed and analyzed with ANALYZE AVW (Biomedical Imaging Resource, Mayo Clinic, http://www.mayo.edu/bir/Software/Analyze/Analyze.html). Individual thylakoids, pyrenoids, and chloroplast membranes were traced on the electron tomographic reconstruction using the program XVOXTRACE. The resolution of the organelles was estimated to be ~10 nm (based on detectability of features and pixel sampling criteria). All computations and graphics were performed on either Silicon Graphics or Sun workstations.