For the reconstructed 3-Dimensional volumes from soft x-ray tomography, we used two methods to visualize organelles. The most basic method is a grayscale image of a one-voxel-thick slice through the volume, with the gray value of each voxel corresponding to the soft x-ray absorption coefficient of the material. show this type of visualization for four cells (including in a mother and daughter that have not yet separated, ). In we display the boundaries between structures that have been digitally segmented from each other. In addition to the plasma membrane of the cells, these images show the borders of organelles within the cell.
Figure 1 Cells imaged by soft x-ray tomography. a–c: One-voxel-thick slices through the reconstructed volumes of four cells (including mother and daughter cells which have not separated), with the gray value of each voxel corresponding to the x-ray absorption (more ...)
(Supplementary Movies 1
are available online. In them the cells in rotate, and the different organelles are shown one color at a time, to better indicate the distribution of organelles.)
In total, we segmented 50–100 organelles in each cell. We included only organelles with diameters of at least 100 nm, approximately twice the observed spatial resolution in the reconstructions. lists the dimensions and volumes of the organelles and cells shown in . The cells varied from 4.0 to 6.6 µm long, with volumes of 17 to 31 µm3. Despite this variation in size, the volume of the nucleus in these cells appeared to comprise between 4 and 6% of the total cellular volume, and the cumulative volume of other segmented organelles was between 14 and 16% of the total cellular volume. Many of these statistics were based on boundaries that were hand-drawn around the organelles. We subsequently used digital segmentation (boundary drawing) of some of the features multiple times to determine the accuracy with which the boundaries had been drawn, and found that the relative standard deviation was less than 5%.
Dimensions and volumes of selected organelles and cells from .
As mentioned in the Materials and Methods section
, by collecting images with the sample in an out of the field, we quantitatively measure projections of the absorption coefficients of our sample, and can then determine these three dimensional absorption coefficients. The values of the voxels in our reconstructed volume thus have value not only because of their contrast with respect to each other, but also because of their absolute values. Every material has a characteristic absorption coefficient (for example, protein, lipid, carbohydrate, water, or glass), so the measured x-ray absorption coefficient can aid in the identification of organelles by giving some indication of their composition. We determined the average x-ray absorption coefficient of each organelle and divided them into five categories, corresponding to the x-ray absorption coefficients indicated in the figure legend (from least to most x-ray dense, the colors are black, blue, green, yellow, and red). The nuclei, which would have an average x-ray absorption coefficient corresponding to blue on the same color scale, are shown in orange to differentiate them from other organelles. show all of the color-coded organelles, while show the five groups of colored organelles in separate panels so that the distributions can be more easily seen. On each row there is also an image of a cross-sectional slice of an organelle characteristic of that group.
Light microscopy was performed for comparison with soft x-ray tomography images. shows these images of S. pombe under two different conditions. show bright field (a), fluorescence (b, c), and overlay (d) images of log phase yeast in media, where the mitochondria have been labeled with MitoTracker (b) and the vacuoles have been labeled with CMFDA (c). These images show a mitochondrial network that is interconnected through the cell, and show a number of spherical vacuoles in each cell. show the corresponding series of images for yeast in PBS which have entered stationary phase. As described in the introduction, in stationary phase yeast there is a cessation of mitochondrial fission, resulting in unbalanced fusion and the formation of a number of spherical mitochondria. Because the cells are in PBS rather than water, thus maintaining the osmolarity of the environment, the vacuole size and number remain approximately the same in these cells.
Figure 2 Bright field, fluorescence, and overlay images of log phase yeast in media (ad) and stationary phase yeast in PBS (e–h). The mitochondria were fluorescently labeled with MitoTracker (b, f), and the vacuoles were labeled with CMFDA (c, g). Scale (more ...)
Our initial assignment of the organelles seen in the x-ray tomography images was carried out on the basis of the organelle appearance and the soft x-ray linear absorption coefficient of the segmented objects. Mitochondria can be readily identified in the tomograms by their characteristic appearance. Whereas most of the organelles have relatively homogeneous absorption coefficients, mitochondria have a thick outer layer that is highly absorptive, while the interior has much lower absorption coefficients, and is very heterogeneous. A cross-sectional image of a mitochondrion and another organelle are displayed in , respectively, to illustrate the differences between them. We believe the dark border of the organelle in is the mitochondrial double lipid membrane layers, and the highly heterogeneous interior is due to the cristae. show surface visualizations of the mitochondria in each cell (the mitochondria are shown in red).
Figure 3 The same cells as shown in , but in b–d, only the nucleus and organelles identified as mitochondria are shown (compare to ), along with a mitochondrial cross section. The remaining organelles are shown in f–h, along with (more ...)
We identified between 5 and 14 putative mitochondria in each cell, and in each case these mitochondria were distributed throughout the cell and accounted for between 4 and 8% of the cell volume (see ). Because of their heterogeneity, the average absorption of the various mitochondria corresponded to multiple different colors in the scheme of . To show the correspondence between the organelles in and , matching arrows and wedges have been added to these figures, as described in the figure captions.