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Reconstruction
Display image description
Single computed slice of a tomographic volume of a chloroplast contained in a blue green algae cell grown under high light conditions. The pyrenoid is very prominent, although the thylakoid membranes are difficult to resolve.
Full resolution image description
Tar file containing two volumes in Analyze 7.5 format: Obliques75.img/hdr and Phaeo27_91sub.img/hdr. Obliques75 was produced by reslicing Phaeo27_91sub along an oblique angle using the oblique section function of Analyze.
Volume_dimension
684, 693, 75
Volume scale
0.005, 0.005, 0.005
Animation description
Animation through the computed slices of a tomographic volume of a chloroplast contained in a blue green algae cell grown under high light conditions. The pyrenoid is very prominent, although the thylakoid membranes are difficult to resolve.

Image 2D
Display image description
Zero tilt image of a 0.5 um thick section of a blue green algae cell taken with intermediate voltage electron microscopy. Contrast is reversed so that electron dense structures appear bright.
Full resolution image description
Tar file containing full resolution unaligned digitized images in suprim format (.f) along with the fiducial mark file used to align them (.fido) and the file containing a list of the angles used (.ang) and the origin file (.origin)

Segmentation
Display image description
Manual segmentation of the chloroplast outer membrane and pyrenoid membrane using Xvoxtrace 2.7 followed by surfacing using Synu. Thylakoid membranes were difficult to resolve and so were not completely traced.
Segmentation file description
Zipped archive containing the surfaced segmentations of the chloroplast outer membrane and the pyrenoid in Synu format along with the Viewdata file required for viewing with Synu. THe original trace file (.trace) containing the manual contours generated in Xvoxtrace is also included.

License
Attribution Only: This image is licensed under a Creative Commons Attribution License. View License Deed | View Legal Code

CCDB:3432*
Project: P1576
Project name
Chloroplast Ultrastructure of Phaeocystis antarctica in High and Low Light Conditions
Description
The three-dimensional morphological rearrangements for two conditions that mimic light conditions for the Antarctic summer and winter were studied in Phaeocystis antarctica Karsten
Funding agency
National Aeronautics and Space Administration
Leader(s)
Tiffany Moisan
Collaborator(s)
Gina Sosinsky
Casey Buitenhuys
Mark Ellisman
Start date
unspecified
End date
unspecified
 
Experiment
Experiment ID
3363
Title
High light condition
Purpose
To examine thylakoid membrane architecture under conditions of high light
Experimenter(s)
Tiffany Moisan
Microscopy product
Microscopy product ID
3432
Instrument
JEOL 4000EX IVEM
Microscopy type
IVEM
Product type
SINGLE TILT
Image basename
Phaeo27
Subject
Species
algae
Scientific name
Phaeocystis antarctica
Strain
Karsten
Group by
Light level
Treatment
continuous blue light 259 ?mol quanta m-2 s-1
Age class
6-8 generation cultured cells
Tissue section
Microtome
Ultramicrotome
Thickness
0.25 µm
Specimen description
Structure
chloroplast
Cell type
algae
Imaging parameters
Type
Electron microscopy product
Recording medium
film
Magification
15000
Accelerating voltage
400 KEV
Notes
Check the magnification before submitting
Specimen preparation
Protocol used
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.
Imaging product type
Type
Single tilt
Description
singlet_desc
Min range
-60 degrees
Max range
60 degrees
Tilt increment
2 degrees
Notes
Specimen was preirradiated prior to imaging