Imaging cell morphology and physiology using X-rays

Abstract

Morphometric measurements, such as quantifying cell shape, characterizing sub-cellular organization, and probing cell-cell interactions, are fundamental in cell biology and clinical medicine. Until quite recently, the main source of morphometric data on cells has been light- and electron-based microscope images. However, many technological advances have propelled X-ray microscopy into becoming another source of high-quality morphometric information. Here, we review the status of X-ray microscopy as a quantitative biological imaging modality. We also describe the combination of X-ray microscopy data with information from other modalities to generate polychromatic views of biological systems. For example, the amalgamation of molecular localization data, from fluorescence microscopy or spectromicroscopy, with structural information from X-ray tomography. This combination of data from the same specimen generates a more complete picture of the system than that can be obtained by a single microscopy method. Such multimodal combinations greatly enhance our understanding of biology by combining physiological and morphological data to create models that more accurately reflect the complexities of life.

Keywords: X-ray tomography; cell structure; imaging.

Figure 1.

A. Morphological study of the four stages of sickle cell anemia in red blood cells carried out by Soft X-ray tomography [103]. B. Diagrammatic representation of cell division in the yeast S. cerevisiae [111], NZ - nocodazole. C. Soft X-ray tomographic study showing the internal morphological changes that takes place during S. cerevisiae cell division. Scale bar = 2 μm [77]. D. Example of quantitative morphological information obtained using soft X-ray tomography. Quantification of nuclear surface areas as a function of cell size of wild type (WT) and mutated (cdc5-nf) cells. This mutation results in ‘nuclear flares’ that appear in cells with a volume of 140 μm³ (black triangle, left hand plot). The plot on the right quantifies nuclear volume as a function of cell size, showing there is no difference in this ratio between wild-type and mutant cells [111].

Figure 2.

A. Soft X-ray analysis of chromatin masses in the nuclei of three types of olfactory epithelium cells, namely multipotent stem cells, neuronal progenitors, and terminally differentiated neurons, together with similar data from HP1β knockout (KO) cells [117]. This morphological study revealed a surprising degree of connectivity exists between chromatin and this persists throughout development and differentiation. Scale bar = 2 μm. B. Alteration of the endoplasmic reticulum-mitochondria interface in Hepatitis C virus (HCV) replicating cells [78]. Comparative analysis of the endoplasmic reticulum-mitochondria topological relationship of vitrified control (A), HCVtcp-infected (B), or HCV replicon-bearing cells (C). Volumes slices, manually segmented surface representation, and surface views of the different areas of interest are shown. Yellow arrows in (A) mark mitochondrial cristae. Two types of abnormal mitochondria were found class 1 (AbMito1) and class 2 (AbMito2). White and black arrows mark matrix condensation arrows and cristae swelling, respectively. Scale bars 0.5 μm.

Figure 3.

A. Soft X-ray tomographic analysis of endosome size in breast cancer (MCF-7) cells incubated with superparamagnetic iron oxide nanoparticles (SPION) for 0 (CTR), 3, 6, 12 and 24 h [133]. The nucleus is shown in blue. Field of view, 11.5 μm x 11.5 μm. SPIONs are color-coded according to size. B. 3D time-lapse cell tracking during gastrulation of African frog Xenopus laevis [139]. Virtual cut through 3D embryo rendering at stage 11.5 depicting ectoderm (blue), mesoderm (orange) and endoderm (green). Abbreviations: dorsal (D), ventral (V), animal pole (AP), vegetal pole (VP), blastocoel (BLC), Brachet’s cleft (BC), blastocoel roof (BLCR). Magnitude of velocity on sagittal slice for two different times. 3D rendering of the highlighted cell pairs in the archenteron and associated trajectories over period of 30min. 3D Velocity field showing difference in collective and singular cell motion at two various times.

Figure 4.

A. Two-dimensional computer-generated section (typically known as an orthoslice) from a reconstruction of a mouse lymphoblastoid cell expressing MiD51–GFP (green), generated using correlated cryo-fluorescence tomography and soft X-ray tomography [154]. B. The same computer-generated slice from the SXT reconstruction as presented in A without fluorescence overlay. The orange rectangle outlines the area of concentrated MiD51–GFP fluorescence. C. Magnification of the area shown in A and B containing a concentration of MiD51–GFP. White arrowheads indicate positions of ER–mitochondria contact sites. D. Maximum intensity projection of the full 3D SXT reconstruction with the contrast reversed so that features that are low-absorbing are shaded black and features that are highly absorbent are shaded white. E. ER (green) and mitochondria (red) segmented out and overlaid with the reconstruction. F. Surface rendering of segmented cellular features, including the nucleus (orange), lipid droplets (blue), ER (green) and mitochondria (red). G. Three-dimensional cutaway of the SXT-generated reconstruction reveals the same 3D location of the MiD51–GFP fluorescence of that shown in A. H. Detailed view of small ER extensions contacting the mitochondria at the MiD51 foci. Scale bars: 2 μm (A, B, D–F); 400 nm (C); 1 μm (H). I. Localization of elements in the different tissues of the eye, such as lens fiber and retinal pigment epithelium, of a 3-dpf (days post fertilization) zebrafish embryo [168].