Soft X-ray tomography and cryogenic light microscopy: the cool combination in cellular imaging

Abstract

Soft X-ray tomography (SXT) is ideally suited to imaging sub-cellular architecture and organization, particularly in eukaryotic cells. SXT is similar in concept to the well-established medical diagnostic technique computed axial tomography (CAT), except SXT is capable of imaging with a spatial resolution of 50nm, or better. In SXT, cells are imaged using photons from a region of the spectrum known as the 'water window'. This results in quantitative, high-contrast images of intact, fully hydrated cells without the need to use contrast-enhancing agents. The cells that are visualized are in close-to-native, fully functional state. The utility of SXT has recently been enhanced by the development of high numerical aperture cryogenic light microscopy for correlated imaging. This multi-modal approach allows labelled molecules to be localized in the context of a high-resolution 3-D tomographic reconstruction of the cell.

Figure 1

Soft x-ray projection image of cryo-fixed NIH 3T3 cells. In this image the contrast is generated by absorption of ‘water window’ x-ray photons by the carbon and nitrogen in the nuclear and cytoplasmic organelles, and not by the use of metal stains. Consequently, the information obtained by this method is quantitative and represents the specimen in a close-to-native state. Scale bar=5 μm. (from ).

Figure 2

Comparative images of the fission yeast Schizosaccharomyces pombe using light and soft x-rays a) Conventional fluorescence microscopy (vacuoles labeled with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) from Invitrogen) b) Differential Interference Contrast (DIC) light microscopy c) Orthoslices (see glossary) through a soft x-ray tomographic reconstruction d) The reconstruction in c after segmentation. Organelles are identified by their characteristic shape, and measured x-ray Linear Absorption Coefficients (LACs). Key: nucleus, blue; nucleolus, orange; mitochondria, grey; vacuoles, white; lipid-rich vesicles, green. (X-ray tomographic reconstructions are an update of the work outlined in ^, ). Scale bar=1 μm.

Figure 3

X-ray tomography of a lymphocyte (T-cell). (a-d) Orthoslices from the tomographic reconstruction of a cryo-fixed T-cell imaged in a capillary. (e-h) Segmented volumes. (e) Cell surface with numerous filopodial extensions. (f-h) Cut-away views showing the typical highly folded large nucleus (cyan) surrounded by a small rim of cytoplasm (purple). Multiple highly absorbing vesicles (yellow) surround the nucleus (cyan), along with less absorbing structures, including mitochondria (magenta) in the cytoplasm. Chromosomes (salmon) and nuclear bodies (green) are seen in the nucleus. Cell is 8 μm in diameter. Scale bars=1 μm.

Figure 4

Tomographic imaging of the three phenotypes displayed by the yeast Candida albicans. a) Yeast-like cells, b) pseudo-hypha, and c) hypha. Images after segmentation of the major organelles are shown. The measured Linear Absorption Coefficients for each segmented organelle are given in Table 1. The hyphal cell, shown in c has a total length of 47 μm. Five tomographic data sets were collected sequentially along the length of the cell. These were ‘stitched’ together after reconstruction using the software package Amira from Visage Imaging Inc. Key: nucleus, blue; nucleolus, purple; mitochondria, orange; vacuoles, yellow, lipid drops, dark green. Scale bar=1 μm. After figures in (M. Uchida et al., submitted).

Box 1, Figure I

Schematic of the main components in XM-2, a new soft x-ray microscope purpose designed for cellular imaging.

Box 1, Figure II

Scanning electron micrograph of a Fresnel zone plate. The inset shows the outer most zones in close-up.

Box 2, Figure I

A 3-dimensional reconstruction of two yeast cells in a glass capillary (top center) calculated from 2-dimensional projection collected sequentially around a rotation axis (shown at 30° increments).