The cellular landscape by cryo soft X-ray tomography

Abstract

Imaging techniques in structural cell biology are indispensable to understand cell organization and machinery. In this frame, cryo soft X-ray tomography (cryo-SXT), a synchrotron-based imaging technique, is used to analyze the ultrastructure of intact, cryo-preserved cells at nanometric spatial resolution bridging electron microscopy and visible light fluorescence. With their unique interaction with matter and high penetration depth, X-rays are a very useful and complementary source to obtain both high-resolution and quantitative information. In this review, we are elaborating a typical cryo correlative workflow at the Mistral Beamline at the Alba Synchrotron (Spain) with the goal of providing a cartographic description of the cell by cryo-SXT that illustrates the possibilities this technique brings for specific localization of cellular features, organelle organization, and particular events in specific structural cell biology research.

Keywords: Correlative microscopy; Cryo soft X-ray tomography; Organelle atlas; X-ray microscopy.

Fig. 1

Attenuation coefficients of carbon and water. The area between the carbon K edge and oxygen K edge is known as the water window

Fig. 2

a A typical workflow at the Mistral Beamline. Cells are grown on top of TEM grids and vitrified. Once frozen, the grids are checked with visible light. Using a regular fluorescence microscope setup and a cryo-stage, the quality of the sample (cell density, flatness of the grid, vitrification success) is checked and potential areas of interest are selected. If available, high-resolution fluorescence data can be collected as well. After fluorescence imaging, the samples are transferred to the TXM chamber. An on-line fluorescence microscope is used to re-locate the areas previously imaged, and tilt-series are collected from the previously imaged cells. b A mosaic image overlaid with the fluorescent signal coming from a mitochondrial stain obtained by cryo structured illumination microscopy (SIM). c One slice of the reconstructed volume from the yellow box in b. Most of the signal is coming from the mitochondrial membranes, which is the target of the dye. Scale bar: B, 10 μm; C, 2 μm

Fig. 3

An overview of cryo soft X-ray data of mammalian cells (fibroblast and NIH-3T3 cells). The different structures have been labeled as follows: N, nucleus; NMC, nuclear membrane channel; Nu, nucleolus; Mi, mitochondria; He, heterochromatin; G, Golgi apparatus; rER, rough endoplasmic reticulum; sER, smooth endoplasmic reticulum; Fid, fiducial markers; Ld, lipid droplets; V, vesicles; Ly, lysosome; En, endosome; Aut, autophagosome. a A mosaic overview of a square within the grid with a cell of interest. Within the mosaic, areas for acquiring tilt-series are selected. The yellow box represents the field of view of the camera. b One slice of the reconstructed volume from the yellow box shown in a. c, d One slice of a reconstructed volume showing the N of two different cells. In c, two Nu are visible, as well as some NMC. The white arrows aim at pores within the double nuclear membrane. In d, Nu is visible and He structures can be seen close to the nuclear membrane. The white arrowheads point to the double membrane. The black arrows point to a detachment of the outer layer of the nuclear membrane, which is called nuclear blebbing. e,f Two Mi forms we were able to observe. A small and elongated form (e) and some swollen Mi (f). g The two forms of Ld usually appear as filled or with an empty core. h, i ER can be found between other organelles. While rER is elongated and usually easy to find, sER membranes are thinner and appear to be randomly distributed. j The G is usually surrounded by low absorbing V and appears as parallel oriented elongated structures close to the N. k, l, m, n Endocytic vesicles in different stages. Depending of the developmental and metabolic state of the cell, different forms can be found. Scale bars: a 20 μm; b 5 μ; c, d 2 μm; e, f, g, h, i, j 1 μm; k, l, m, n 0.5 μm