Figure 1.
Comparison between images obtained from the surface of epoxide blocks containing red cells and transmission electron microscopy images of thin sections.
(A) Image of an epoxide block surface containing infected erythrocytes obtained at 5 KeV detecting backscattered electrons. Note that membranes and other structures within the cells generate more signal (gray-white regions) in contrast to the dark background. Pixel size was 6.07 nm with an image size of 2048×1768 pixels. (B) Image shown in A after contrast inversion. Note that the contrast aspects of the image are similar to TEM images (C). (D) Backscattered image of a block surface at higher magnification. Infected erythrocytes were imaged at 2 KeV, the pixel size was 3.5 nm with an image size of 1024×943 pixels. (E) Image of an equivalent material obtained by TEM of a thin section. Bars represent 2 µm (A–C) and 500 nm (D–E). iRBC, infected red blood cell; P, parasite; PVM, parasitophorous vacuole membrane; H, hemoglobin filled compartment; MP, membrane profile.
Figure 2.
FIB-SEM reconstruction of multiple red cells simultaneously.
(A–C) Images of the block face where the whole thickness of one cell was milled. (D–I) Reconstruction and modeling of multiple erythrocytes contained in the volume. Bars represent 500 nm (A–F) and 2 µm (G–I).
Table 1.
Morphometric analysis of the structures reconstructed from FIB-SEM images.
Figure 3.
3D reconstruction by FIB-SEM shows surface morphology and internal details of an infected red cell.
(A–B) 3D model of the surface of an infected erythrocyte showing deformations as a consequence of the presence of the parasite. (C) Modeling of parasites located underneath (PVM-yellow) and membranous structures (green). (C-inset) Semi-transparent iRBC, showing that the deformations on the RBC surface coincide with the presence of the parasites. (D, E) Different views showing the three-dimensional organization of the parasites inside a parasitophorous vacuole (PV, yellow), parasite membrane (purple, in D), membranous structures (MS, green), multi-lobular nuclei (D, inset, blue) and hemoglobin-containing tubules (HCT, E, inset, dark red). Bars: A, D and E, 1 µm. B–C, 500 nm.
Figure 4.
3D models of membranous structures and projections of the PVM within the red blood cell.
(A) Membrane profiles delimiting internal spaces in the cytoplasm of an infected erythrocyte. (B) Modeling of each structure (MS, green) showing their three dimensional organization and heterogeneity in shape and size (B–D). (C) Model showing projections of the PVM that may contribute to the formation of a TVN (PVMp, inset). (D) Projections from the PVM visualized in cross section (approx. 10 cross section images) P, parasite. Bars represent 500 nm (A–C), 1 µm (D) and 500 nm (D, inset).
Figure 5.
Modeling of multiple cells reveals a polymorphic aspect of hemoglobin-containing tubules in all parasites.
(A) Vesicular aspect of endosomes in a single image of the block surface (arrows). (B, C) Interconnected tubular aspect of a parasite hemoglobin-containing compartment. (D–M) Polymorphic characteristics of the parasite hemoglobin-containing tubules (HCT) in different cells. Note that such structures appear as a single compartment in all cells analyzed. (N, R) Rotation of a single tubular compartment around the x axis. Bars represent 500 nm (A–C) and 200 nm (D–R).
Figure 6.
Simultaneous visualization of surface and intracellular structures by FIB-SEM.
(A) Scanning electron micrograph of a Plasmodium chabaudi infected erythrocyte. Note the deformations in the erythrocyte membrane due to the presence of the parasite. (B) Transmission electron micrograph of a vesicle derived from the plasma membrane of the erythrocyte. (C, D) Similar vesicles were observed in the serial images obtained with FIB-SEM. (E) 3D model of an infected-erythrocyte showing similar deformations seen on the surface of the erythrocyte membrane by field emission scanning electron microscopy (A). (E-inset) Internal aspect of the membrane invaginations seen from the external perspective in E. (F) Visualization of inner portions of the erythrocyte showing vesicles of different sizes, presumably originated from membrane invaginations. Bars represent 500 nm (A), 200 nm (B), 500 nm (C), 500 nm (D), 500 nm (E), 500 nm (F) and 250 nm (E, inset).
Figure 7.
Schematic model summarizing the structure of Plasmodium chabaudi infected red blood cell.
(iRBC) Infected red blood cell; (I) Invaginations of the plasma membrane of the iRBC; (V) Vesicles distributed along the cytoplasm of the iRBC; (MS) Membranous structures enclosing the hemoglobin; (PVMp) Projections of the PVM; (P) Parasite; (HCT) Hemoglobin-containing tubules (only closed tubules are presented); (N) Nucleus.