Quantitative 3-D imaging of eukaryotic cells using soft X-ray tomography

Abstract

Imaging has long been one of the principal techniques used in biological and biomedical research. Indeed, the field of cell biology grew out of the first electron microscopy images of organelles in a cell. Since this landmark event, much work has been carried out to image and classify the organelles in eukaryotic cells using electron microscopy. Fluorescently labeled organelles can now be tracked in live cells, and recently, powerful light microscope techniques have pushed the limit of optical resolution to image single molecules. In this paper we describe the use of soft x-ray tomography, a new tool for quantitative imaging of organelle structure and distribution in whole, fully-hydrated eukaryotic Schizosaccharomyces pombe cells. In addition to imaging intact cells, soft x-ray tomography has the advantage of not requiring the use of any staining or fixation protocols-cells are simply transferred from their growth environment to a sample holder and immediately cryofixed. In this way the cells can be imaged in a near native state. Soft x-ray tomography is also capable of imaging relatively large numbers of cells in a short period of time, and is therefore a technique that has the potential to produce information on organelle morphology from statistically significant numbers of cells.

The analysis of microscopy imaging on liquid crystalline components of the cell nucleus

“…Osmium tetraoxide-fixed nuclei may resist radiation damage during XCT data collectionas no significant shrinkage was observed and the condensed nucleus size calculated by synchrotron-based hard X-ray tomography was close to the size measured using CLSM. The image contrast of three-dimensional bioimaging of nuclei using XCT is very low because chromatins have relatively homogenous absorption efficiencies unlike entire eukaryotic cell, such as yeast, it's based on the different absorption efficiencies of the natural organic compositions of different subcellular organelles [16,40]. Consequently, heavy metal staining was used to enhance the contrast, which is frequently used for TEM.…”

“…Stimulated emission depletion (STED) microscopy provides 3D images of biological structures with high spatial resolution [15]. However, all of the UV confocal microscopy techniques are limited in that only fluorescently labeled targets can be imaged, and unlabeled targets cannot be visualized [12,14,16].…”

“…Synchrotron radiation-based X-ray tomography can traverse the sample thickness limits of electron microscopy and resolution restrictions of CLSM [12,17,18]. Several groups demonstrated high resolution 3D imaging of biological objects of yeast [16,17,19], lymphocyte [18], virus membranes [20], bacteria [21,22], eukaryotic chromosomes [23] and algae [24] in the soft X-ray in the "water window" spectral region (wavelength region: 2.34 nm -4.44 nm, photon energy region: 0.28 -0.53 KeV) [16,17,20] and hard X-ray spectral region (wavelength region: 0.01 nm -0.62 nm, photon energy region: 2 -100 KeV) [21,23]. Compared with soft X-ray microscopy, hard X-ray microscopy has relatively large penetration depths, focal lengths and depths of focus, which has advantages for 3D reconstruction of much thicker cells and tissues [25,26].…”

The Best of Both Worlds: 3D X-ray Microscopy with Ultra-high Resolution and a Large Field of View

“…Resolution for 2D imaging down to 12-30 nm has been recently reported [1][2][3][4]; meanwhile 3D resolution to 30-60 nm and below is also now widely available [5][6][7]. This nano-CT technique has demonstrated many advantages in studying the 3D structures of complex samples at various length scales and has satisfied applications in a wide range of fields, such as material science [8][9], cellular biology [10][11][12], solid oxide fuel cells [13][14], and environmental science [15][16][17].…”

3D Chemical Imaging with STXM Spectro-Tomography

“…Soft X-ray tomography using full field X-ray transmission microscopy [1,2] or cryo-STXM [3] has been performed at only one energy which provides density/thickness contrast with limited and potentially ambiguous chemical information. In contrast, spectro-tomography [4-6] acquires tomograms at multiple photon energies to provide true, quantifiable 3D chemical mapping through element-and species-specific, near edge X-ray absorption (NEXAFS) contrast of the components.…”