Figure 1.
Soft x-ray dataset of Chlamydomonas reinhardtii wild type and cw15+ strain used for tomographic reconstruction.
(A) Wild type strain tilt-series taken from −60° to +60° and 0° image after acquisition. Low tilt angles were exposed for 2 s, higher tilt (40° onwards) for 8 s. All angles provide good structural resolution and were all used for the reconstruction of the tomograms. Bar, 2 µm. (B) cw15+ strain tilt-series −60° to +60° and 0° image after acquisition were taken as above. Bar, 2 µm.
Figure 2.
Tomographic reconstruction using IMOD, Bsoft and Alignator reconstruction software applied to the Chlamydomonas cw15+ dataset shown in Fig. 1B.
(A) Representative tomographic sections using the fiducial based Etomo package included in IMOD. Bar, 2 µm. (B) Representative tomographic sections using the back-projection based alignment procedure Bsoft. (C) Representative tomographic sections using the fiducial less alignment approach Alignator. (D) Z-section generated with Alignator. A number of organelles can be identified: Chloroplast (Cp) with the starch containing pyrenoid (Py) and thylakoids (Th); nucleus (Nc) with nucleolus (Ncl); vacuoles (Va); mitochondria (Mt); lipid bodies (Li). Bar, 2 µm.
Figure 3.
Segmentation of cellular structures in Chlamydomonas cells.
(A) Segmentation of a wild type cell tomogram. Top, stereo pairs in front view and after 180° rotation. Bottom, from left to right: chloroplast with pyrenoid; mitochondria; flagella; nucleus with nucleolus; vacuoles; lipid bodies. Bars, 2 µm. The tomogram and segmented volumes of the same cell are shown in Videos S4 and S5 (B) Segmentation of a cw15+ cell tomogram. Segmented volumes are presented as in A. The tomogram and segmented volumes of the same cell are shown in Videos S3 and S6.
Figure 4.
Ultrastructural details in wild type and cw15+ cell tomograms.
(A) Sections of flagella in the tomogram of the wild type cell. The arrow indicates a microtubule doublet. Bar, 1 µm. Inset: enlarged view showing flagellar membrane and microtubule doublets; bar, 200 nm. (B) Basal apparatus with distal connecting fiber (arrow) connecting the two basal bodies in the wild type cell. Pm, plasma membrane; Cw, cell wall. Bar, 1 µm. (C) Nuclear envelope membranes in the tomogram of the wild type cell. Bar, 1 µm. Inset: enlarged view showing inner and outer nuclear envelope membrane; bar, 200 nm. (D) Nuclear envelope membranes in the tomogram of the cw15+ cell. Bar, 1 µm. Inset: enlarged view showing inner and outer nuclear envelope membrane; bar, 200 nm. (E) Putative ER membrane (arrow) emanating from the nuclear envelope of the cw15+ cell. Bar, 500 nm. (F) Contrast rich cisternae structure (black arrow) which could be part of the Golgi apparatus in the cw15+ cell. Bar, 500 nm. Inset, possible vesicle budding site (white arrow). Bar, 200 nm. (G) Ultrastructural details of the chloroplast of the cw15+ cell: thylakoids and granae (arrow) and pyrenoid (Py). Bar, 1 µm. (H) Thylakoids and plastid envelope membranes of the chloroplast of the cw15+ cell. Bar, 500 nm.