Cryo‐Soft X‐ray Tomography of the Cell

Abstract

Cryo soft X‐ray tomography (cryo‐SXT) is an emerging X‐ray microscopy technique for three‐dimensional visualisation of cryo‐preserved, whole, unstained cells at a spatial resolution of 30 nm (half‐pitch). It is, therefore, bridging the gap between the low‐resolution visible light imaging techniques that provide dynamic information of specific processes, and the high resolution electron microscopy ones which achieve molecular resolution. As a first‐order approximation, in SXT as in electron tomography, a series of absorption contrast projections of the specimen are collected at different angles to finally compute the 3D absorption maps. In addition to the 3D structural information that can be obtained, the chemical sensitivity of the X‐rays to the atomic elements of the sample can also be exploited to enhance visualisation of these elements or to distinguish different chemical compositions, as well as to extract quantitative information from the data.

Key Concepts:

  • Tomography allows three‐dimensional visualisation of the sample.

  • The soft X‐ray water window energy range allows penetrating up to 10 μm of water therefore enabling a full cell to be imaged.

  • Cryo‐fixation is the only sample preparation step required in soft X‐ray tomography (SXT).

  • Cryo soft X‐ray tomography data suffers from the limited depth of field of the Fresnel zone plate objective lens which is usually smaller than the thickness of vitreous ice that can be penetrated.

  • The interpretation of soft X‐ray tomography data is complex and requires complementary information from electron and optical microscopy.

Keywords: tomography; transmission X‐ray microscope; water window energy range; cryo‐fixation; cellular imaging

Figure 1.

(a) Penetration power of soft X‐rays in the water window energy range, between the absorption edges of carbon (284 eV) and oxygen (543 eV). At 520 eV, usual working energy, 10 μm of vitrified water can be penetrated. (b) Comparison of thickness ranges and spatial resolutions along the optical axis of cellular imaging techniques without staining or chemical fixation, such as cryo‐electron tomography (CET), cryo‐scanning transmission electron tomography (CSTET) (Greyer Wolf et al., ), cryo‐soft X‐ray tomography (SXT), cryo‐FIB scanning electron microscopy (Schertel et al., ), super‐resolution optical microscopy and confocal microscopy. (c) Workflow of the different sample checking steps before a cryo‐SXT experiment. First, the samples are checked in vivo by fluorescence microscopy to assess sample preparation. Second, the sample are vitrified and subsequently screened again at cryogenic conditions. Fluorescence maps are produced to localise relevant cells of features inside cells. Finally, the samples are transferred to the TXM chamber at the synchrotron beamline. The TXM parts are shown in the last sketch: a condenser glass capillary of 100 mm length and a focal length of 10 mm focus the light onto the sample which is rotated to collect the tilt angle projections, and a FZP lens of 150 μm and a focal length of 2.5 mm forms the image of the sample onto the CCD detector with a magnification of about 1000–1500. An on‐line optical microscope allows relocating the relevant cells or target cellular features which will be imaged with X‐rays after the sample is transferred to the TXM chamber.

Figure 2.

Example of correlative microscopy investigation of Ptk2 cells infected with a GFP‐expressing vaccinia virus strain (courtesy of J. L. Carrascosa). (a) and (b) In vivo light microscopy of the infected cells growing on holey carbon grid. (a) Epifluorescent image. DAPI, in blue, labels the DNA, in red WGA labels membranes of the cell and GFP in green is expressed only in infected cells allowing localising which cells have been nicely infected among the cells grown on the grid. (c) Cryo light microscopy image on‐line with the transmission X‐ray microscope at ALBA of the same area in (a) and (b). (d) 2D soft X‐ray cryo projections of the same square area of the grid. The yellow arrows and discontinuous lines point to the position of the same infected cell from (a) to (d). (e) The 0° soft X‐ray cryo projection raw image of the same cell at higher magnification. (f) Virtual slice of the reconstructed soft X‐ray cryo‐tomogram xy plane. N marks the position of the cell nucleus. (g) & (h) Segmentation showing the nucleus and the location of the different viral forms (red are mature virions and yellow are immature ones). SXT data were collected at the Mistral beamline at ALBA.

Figure 3.

(a) Isolated vaccinia virus cryo‐SXT subvolumes from the work of Carrascosa et al. () are shown. The high contrast achieved with soft X‐rays allowed visualising membranes and inner compartments of the virus with a resolution of 25.7 nm (half‐pitch), 3.8 times less than with cryo ET but with volume thickness 10 times larger. Scale bare: 300 nm. Data collected at the TXM‐U41 at HZB‐Bessy II (reproduced from Carrascosa et al., © Elsevier). (b) SXT 0° projection of a whole vitrified cell in which nucleus, nucleoli, cytoplasm and plasma membrane are clearly visible (courtesy of J.L. Carrascosa). Data collected at the TXM‐U41 at HZB‐Bessy II. (c) Cryo‐SXT and EM comparison of mitochondria morphology of different type of cells, PtK2, MCF7, T cells and HuH‐7 (courtesy of J. L. Carrascosa (Ptk2 and MCF7), E. Veiga and P. Gastaminza unpublished data, respectively). Note that the X‐ray data shown are extracted from volumes of about 3–4 μm of cytoplasm while EM data are derived from thin sections of 50 nm. Data collected at TXM‐U41 at HZB‐Bessy II (PtK2, MCF and T cells) and at Mistral beamline at ALBA (HuH‐7).

Figure 4.

(a) Cryo‐SXT virtual slice of the cellular contact of a T cell (TC) and an antigen presenting cell (APC) in which the ultrastructure of the immunological synapse area (Calabia‐Linares et al., ) is clearly visible (courtesy of E. Veiga, unpublished data). Scale bar represents 2 μm. Data collected at the TXM‐U41 at HZB‐Bessy II. (b) Rendering visualisation of the reconstructed volumes of the two cells.

Figure 5.

As demonstrated in the work of Cruz‐Adalia et al. (), bacteria trans‐infect T cells through the immunological synapse (Cruz‐Adalia et al., ). (a) Virtual slice of a tomogram showing an infected dendritic cell (DC) exposing internal bacteria near the immune synapse (IS) with a T cell (T). N labels the nucleus position of the T cell and V some vesicles. Bacteria are visible in the dashed orange square. (b) Consecutive virtual slices every 460 nm showing the proximity of the three bacteria, in the orange square of A, to the IS with a T cell. Scale bars in (a) and (b) represent 2 μm. (c), (d), and (e) Volumetric representations of the tomogram in (a) and (b). The T cell is represented in cyan and its nucleus is shown in blue. The dendritic cells (DC) are shown in grey and the bacteria in red. Data collected at the Mistral beamline at ALBA.

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Pereiro, Eva, and Chichón, Francisco J(Nov 2014) Cryo‐Soft X‐ray Tomography of the Cell. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0025582]