Journal of Microscopy, Vol. 255, Issue 2 2014, pp. 65–70
doi: 10.1111/jmi.12139
Received 13 February 2014; accepted 22 April 2014
Biological applications of cryo-soft X-ray tomography E . D U K E ∗, K . D E N T ∗, M . R A Z I † & L . M . C O L L I N S O N ‡
∗ Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire, UK
†Secretory Pathways Laboratory, London Research Institute, Cancer Research UK, London, UK ‡Electron Microscopy Unit, London Research Institute, Cancer Research UK, London, UK
Key words. Cells, CLXM, Correlative, Cryo-soft X-ray tomography, Cryo-fluorescence, Cryo-preservation, Cryo-SXT, Organelles, Synchrotron.
Summary X-rays are used for imaging many different types of biological specimen, ranging from live organisms to the individual cells and proteins from which they are made. The level of detail achieved as a result of the imaging varies depending on both the sample and the technique used. One of the most recent technical developments in X-ray imaging is that of the soft X-ray microscope, designed to allow the internal structure of individual biological cells to be explored. With a field of view of 10–20 × 10–20 μm, a penetration depth of 10 μm and a resolution of 40 nm3 , the soft X-ray microscope neatly fits between the imaging capabilities of light and electron microscopes.
X-rays for imaging soft biological tissues Soft X-ray tomography (SXT) is similar in concept to electron tomography (Lucic et al., 2013). In SXT, the sample is tilted during image acquisition, and the resulting tilt series is reconstructed to give a stack of virtual images through the volume of the sample. The term ‘soft’ is used to differentiate the X-rays from ‘hard’ X-rays, which have a higher energy (>5–10 keV) and are able to penetrate into heavier materials such as bone and metals. Cells have very little contrast in hard X-ray microscopes, because low atomic number elements (carbon, oxygen, hydrogen, nitrogen) do not absorb high energy X-rays well. However, ‘soft’ X-rays with energy of 500 eV are preferentially absorbed by carbon rather than water (oxygen), and are able to penetrate through a depth of 10 μm of carbon/water. Given the nature of soft X-rays, a key aspect of most SXT microscopes is that the entire system is in vacuum. Thus, the cells must be embedded within a support matrix. Vitreous ice is the obvious choice, due to its low absorption of the X-rays. In addition, cryogenic temperatures reduce the ability of the X-rays to damage the samples during data collection. Thus, a unique Correspondence to: L. M. Collinson, Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK. Tel: +44 (0) 207 269 3416; e-mail: [email protected]
C 2014 The Authors C 2014 Royal Microscopical Society Journal of Microscopy
property of the cryo-SXT microscope is that 3D ultrastructural information is obtainable from intact, unstained biological cells where the only sample preparation process has been cryo-preservation.
The cryo-soft X-ray microscope The cryo-SXT microscope is broadly similar to any other microscope, with certain parts tailored to the specific requirements of the X-rays used. The key to the success of the technique is the provision of a stable source of 500 eV X-rays. There are two alternatives for this – a synchrotron facility or a plasma source. The synchrotron has many advantages – it is intense so exposure times per image are short (1 s) and the energy of the X-rays can be varied, allowing compositional information to be collected via exploitation of elemental absorption edges. However, access to synchrotron facilities can be limited, and the time between proposal submission and actual beamtime (assuming successful application) can be long. The alternative, a nitrogen plasma source, has been found to work exceptionally well though data acquisition times are significantly longer than at the synchrotron (many hours compared with 10–30 min on the beamline). There are currently four cryo-SXT microscopes installed, operational and actively studying biological samples at synchrotrons worldwide, including the Advanced Light Source (ALS) in Berkeley (CA, USA) (Le Gros et al., 2005), BESSY II in Berlin (Germany) (Schneider et al., 2012) and ALBA in Barcelona (Spain) (Pereiro et al., 2009). Here, we focus on the newest of the microscopes, installed at the Diamond Light Source in Didcot (UK) (Fig. 1A) (Carzaniga et al., 2013). This cryo-SXT microscope (Xradia, now Carl Zeiss X-ray Microscopy Inc, Pleasanton, CA, USA) is currently operating with a nitrogen plasma source (Energetiq Technology Inc, Woburn, MA, USA), and is due to be connected to the synchrotron X-ray source in late 2014. The soft X-rays (from the synchrotron or plasma source) are focused onto the sample using a condenser (Fig. 1B). Once the X-rays have passed through the sample, they are then collected
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Fig. 1. Cryo-SXT microscope at the Diamond Light Source. (A) The cryo-SXT microscope (Xradia, now Carl Zeiss X-ray Microscopy Inc) is currently operating in off-line mode using a nitrogen plasma source (Energetiq). The microscope will move to a synchrotron X-ray source in 2014. (B) The main components inside the microscope vessel, including the capillary condenser (CC); sample cartridge containing a 3 mm grid mounted at the data collection position (SC); zone plate holder with the zone plate mounted at the tip (ZP); cryoshield (CS) and visible light microscope (VLM) objective.
and refocused onto the detector using the objective lens. For the soft X-ray microscope, the condenser is either a capillary lens or a zone plate and the objective is a zone plate. A zone plate is a series of concentric rings (zones) of alternating transparent and opaque material from which the X-rays diffract to form an image at the focal plane. It is the radius of the outermost ring of the zone plate that governs the theoretical resolution of the microscope. At present, the highest resolution zone plate in routine use for tomography of biological samples is 25 nm. The higher the resolution of the zone plate, the shorter its focal length. At 25 nm the zone plate must be placed 1–2 mm from the sample. Given that data collection requires sample rotation this can place a limit on the achievable angles, though this does vary depending on the type of sample mount used. In reality, the resolution of a data set is also limited by other factors including sample preparation, sample thickness and data reconstruction techniques. Preparing samples As with all imaging techniques, sample preparation is key to collecting high-quality data. The technique for sample preparation is dependent on the microscope chosen for data collection. In the current cryo-SXT facilities, samples are mounted using either capillaries or electron microscopes (EM) grids. Ultrathin-walled capillaries, used in the ALS microscope, are made by heating and pulling narrow-walled small diameter quartz capillary tubes such that their final internal diameter is approximately equal to the size of the sample being studied
(in the order of a few microns). The capillaries are then filled with a solution containing the samples in question and plunge frozen. This method lends itself particularly well to smaller cells that grow in suspension, such as bacteria and yeast. The BESSY II microscope currently requires bespoke EM grids designed specifically to allow sample rotation without colliding with the zone plate, so called ‘high tilt’ or ‘HZB-2’ grids, whereas the ALBA and Diamond microscopes can accept standard 3 mm transmission electron microscopy (TEM) grids. For both grid types, suspensions can be applied to the grid or adherent cells can be grown on grids that have been coated in a thin layer of holey carbon (Fig. 2A). It is important to have low confluency in order that individual cells can be viewed without overlap as the sample is tilted. Cells can be mapped prior to cryo-fixation using confocal microscopy, so that confluency can be assessed and cells expressing fluorophores located for correlative imaging (Fig. 2B). Plunge freezing in liquid nitrogen-cooled liquid ethane is currently the most popular method of cryo-preservation. This enables rapid vitrification and avoids the formation of hexagonal ice crystals that would otherwise disrupt cellular ultrastructure (Hagen et al., 2012). Immediately prior to freezing, fiducial markers are added to the grid to aid post-acquisition alignment of the tilt series (Fig. 2C). Grids are then blotted, another key step in obtaining high quality data (Fig. 2D). Over blotting before plunging can result in dehydration of cells and susceptibility to X-ray damage; under blotting leads to thick ice, resulting in poor penetration of the X-rays and weak contrast in the final image stack (particularly at high tilt angles). C 2014 The Authors C 2014 Royal Microscopical Society, 255, 65–70 Journal of Microscopy
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Fig. 2. Sample preparation workflow for cryo-SXT of whole mammalian cells. (A) Cells are grown on 3 mm finder grids (Diamond, ALBA) or HZB-2 grids (BESSY II). (B) Grids are screened for confluency and for cells expressing fluorophores in the confocal microscope for correlative experiments. (C) Gold fiducial markers are added to the grid to aid post-acquisition data alignment. (D) The grid is blotted to remove excess liquid, and (E) plunge frozen in liquid nitrogen-cooled liquid ethane. (F) An additional screening step can be included to map cells of interest and assess ice quality. (G) Frozen Hek293 cells with a fluorescent label highlighting mitochondria. (H) Grids are sent to the synchrotron beamline in a dry nitrogen shipper. Scale bars – B: 100 μm, G: 20 μm. C 2014 The Authors C 2014 Royal Microscopical Society, 255, 65–70 Journal of Microscopy
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The grids are plunge frozen (Fig. 2E), and transferred to cryostorage under liquid nitrogen. A cryo-fluorescence screening step can be used in the home laboratory (Fig. 2F) to locate cells of interest (Fig. 2G) and screen for artifacts. This step is critical for correlative imaging if the cryo-SXT microscope does not have an integrated fluorescence microscope (currently the case at ALBA), and can also help to maximise throughput at beamlines with integrated fluorescence microscopes (BESSY, Diamond). The grids are then shipped to the synchrotron for imaging (Fig. 2H). Imaging with a cryo-SXT microscope Cryo-SXT has benefited greatly from technical developments that have taken place in both EM and X-ray crystallography. Assuming a microscope that accepts samples grown on EM grids, then the mechanism for mounting the grids into the microscope takes much of its technology from cryo-TEM. Grids are mounted in holders and loaded into the microscope via a vacuum load-locked transfer device, which allows transfer of the samples into the microscope without needing to vent the microscope to atmospheric pressure. At beamlines equipped with an integrated visible light microscope with fluorescence capability, it is possible to further screen the grid after insertion into the cryo-SXT microscope to identify cells that are suitable for data collection. Once a cell has been selected, it is placed at the centre of sample rotation, which also coincides with the focal position of both the capillary condenser and zone plate objective lenses. Data acquisition proceeds via collection of a series of images at angular tilts, usually in the range of ±70°, a restriction imposed by the close proximity of the zone plate. At this point we term the data collected a tilt series. A strategy that takes into account the increased sample thickness at high angles when setting exposure times at each tilt has been found to be beneficial to final tomogram quality. Care must also be taken to ensure that the sample is not destroyed by the X-rays part-way through the data collection. At present, the procedure established to assess the damage during data collection is a comparison ‘by eye’ of the images taken at 0° before and after the tilt series acquisition. The images themselves are collected on a direct detection, back thinned CCD detector.
fiducial markers. The quality of this initial alignment has a huge influence on the quality of the sinogram that is calculated, and thus the feature resolution of the final tomogram. The reconstruction is then performed, usually with a back projection or iterative reconstruction algorithm. This leads to a stack of virtual images with high spatial resolution (25–40 nm3 ) through the volume of the cell. Contrast of cellular membranes in the tomograms resembles that of TEM, even though no heavy metal stains are added to the cells (Fig. 3). Indeed, contrast is directly related to the composition of the cellular structures, raising the possibility of automatic recognition of cellular structures by their density alone, measured as the linear absorption coefficient (LAC) (Parkinson et al., 2008). In the case where a complete rotation range can be collected (at the ALS using capillaries), it is possible to automatically segment organelles in the tomogram using the LAC. In the case of a limited tilt series (BESSY, ALBA, Diamond), the relationship of the LAC to cellular membranes is more complex, and automation of the segmentation process is less straightforward. Finding structures of interest using cryo-SXT A number of cellular structures can be recognised in soft Xray tomograms by their morphology alone, based on prior information from electron microscopy. The cell nucleus, nuclear envelope, plasma membrane, mitochondria and endoplasmic reticulum (ER) are easily discernible and can be followed through sequential images in the tomogram for 3D analysis (Fig. 3A,B and Movies S1,S2). Resolution is sufficient to see cristae in mitochondria, the lumen of the ER (Fig. 3C), the space between the two lipid bilayers of the nuclear envelope and nuclear pore complexes (Duke et al., 2013). An atlas of these and other organelles with less characteristic morphologies is gradually being compiled by the cryo-SXT community (Muller et al., 2012), including endosomes (Fig. 3D) identified using gold particles targeted to internalised receptors (Fig. 3E) (Duke et al., 2013). Features of interest may also be identified using fluorescent markers and a correlative workflow (Fig. 2), which has so far proved valuable in the study of mitochondria in yeast (Parkinson et al., 2008), virus-induced vesicular structures (Hagen et al., 2012) and autophagy (Duke et al., 2013) in mammalian cells.
Reconstructing data To date, cryo-SXT tilt series have been reconstructed into tomograms using a range of software packages, all originally developed for EM tomography. Therefore, at present, no account is taken of the properties of the X-rays (as opposed to electrons) or that the depth of focus of the X-rays is typically less than the sample thickness (for mammalian cells). Despite this, the results are quite spectacular. To create the tomogram from the tilt series, the background corrected images are first aligned to each other using the gold
The cryo-SXT niche A cryo-soft X-ray tomogram allows the scientist to build up a 3D image of the near-native state cell (Fig. 3F,G), which shows with stunning clarity the morphology, interactions and complexities of subcellular organelles. Volumetric analysis of cells and organelles is more accurate than light microscopy due to the resolution of the technique, and more accurate than electron microscopy due to the absence of shrinkage artifacts (Uchida et al., 2011). The absence of heavy metal C 2014 The Authors C 2014 Royal Microscopical Society, 255, 65–70 Journal of Microscopy
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Fig. 3. Ultrastructure of whole, unstained Hek293 cells imaged by cryo-SXT at the Diamond Light Source. (A)–(D) Virtual slices from tomograms collected using the cryo-SXT microscope at the Diamond Light Source, currently operating with a nitrogen plasma source. Tilt series were collected from ±60° and the resulting images were reconstructed into a tomogram using the SIRT algorithm in IMOD (http://bio3d.colorado.edu/imod). (A) Three image planes from a tomoX stack, moving from the bottom (left panel) to the top (right panel) of the cell. At the bottom of the cell (left panel), the holes in the QuantifoilTM carbon support are visible (dashed circles) as well as the 250 nm gold fiducial markers (arrowheads). Endoplasmic reticulum (ER) and mitochondrial networks (m) with fine connections (arrows) can be followed through the stack. See also Movie S1. (B) As in (A), but also showing the plasma membrane (PM). See also Movie S2. (C)–(E) Ultrastructure is similar to that seen in TEM, even though no heavy metal stains are used. (C) Cristae can be seen inside mitochondria (m) and the lumen of the endoplasmic reticulum (ER) is visible. (D) Endosomes with tubular extensions are visible, identified by comparison with, (E) Transferrin-receptor-gold loaded endosomes imaged by cryo-SXT at the U41-TXM microscope at BESSYII in Berlin (Duke et al., 2013). (F) and (G) XYZ planes showing the sample thickness and three dimensional nature of the data from the tomogram in panel A (F) and B (G) (screenshots from IMOD). Scale bars – 2 μm. C 2014 The Authors C 2014 Royal Microscopical Society, 255, 65–70 Journal of Microscopy
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stains allows detection of compositional information, used to great effect in analysis of iron-containing red blood cells infected with the malaria parasite P. falciparum (Hanssen et al., 2012). Fine connections can be seen between individual mitochondria that are not easily visible by either light or electron microscopy (Fig. 3) (Duke et al., 2013) enabling studies of mitochondrial networks in health and disease states. Volatile structures that are prone to chemical fixation artifacts, such as tubular endosomes and autophagosomes, are optimally captured by cryo-preservation and can be analysed in 3D through the image stack (Duke et al., 2013), with wider applications to studies of organelle biogenesis and membrane trafficking. Though cryo-SXT may currently lack the resolution to place macromolecular structures into cellular context, there is already a proven niche for cryo-SXT in the analysis of the near native-state cell. It is still early days in the development of the cryo-SXT field, with technological advances in zone plates, correlative workflows and tailored software likely to drive forward the scope of scientific applications. Indeed, there has been a shift in publications from development of the technology and proof-of-principle studies to incorporation as an imaging tool in bioscience investigations. With increasing uptake from scientists in collaboration with imaging experts, it would be unwise to underestimate the potential of this fledgling imaging technique.
Acknowledgements We gratefully acknowledge funding from Cancer Research UK (MR & LC). The research leading to the results shown in Figure 3(E) received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570). ED and KCD would like to thank the Xradia team for their outstanding dedication to the development of the soft X-ray microscope and to Energetiq for their support in the implementation of the plasma source. We would also like to thank Dr Raffaella Carzaniga, Dr Marie-Charlotte Domart and Elzbieta Kubiak-Goswami for the photographs in Figure 2.
References Carzaniga, R., Domart, M.C., Collinson, L. M. & Duke, E. (2013) Cryo-soft X-ray tomography: a journey into the world of the native-state cell. Protoplasma 251, 449–458. Duke, E.M.H., Razi, M., Weston, A., et al. (2013) Imaging endosomes and autophagosomes in whole mammalian cells using correla-
tive cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM). Ultramicroscopy. Epub ahead of print. doi: 10.1016/j.ultramic. 2013.10.006 Hagen, C., Guttmann, P., Klupp, B., Werner, S., Rehbein, S., Mettenleiter, T.C., Schneider, G. & Grunewald, K. (2012) Correlative VISfluorescence and soft X-ray cryo-microscopy/tomography of adherent cells. J. Struct. Biol. 177, 193–201. Hanssen, E., Knoechel, C., Dearnley, M., Dixon, M.W., Le Gros, M., Larabell, C. & Tilley, L. (2012) Soft X-ray microscopy analysis of cell volume and hemoglobin content in erythrocytes infected with asexual and sexual stages of Plasmodium falciparum. J. Struct. Biol. 177, 224–232. Le Gros, M.A., McDermott, G. & Larabell, C.A. (2005) X-ray tomography of whole cells. Curr. Opin. Struct. Biol. 15, 593–600. Lucic, V., Rigort, A. & Baumeister, W. (2013) Cryo-electron tomography: the challenge of doing structural biology in situ. J. Cell. Biol. 202, 407– 419. Muller, W.G., Heymann, J.B., Nagashima, K., Guttmann, P., Werner, S., Rehbein, S., Schneider, G. & McNally, J.G. (2012) Towards an atlas of mammalian cell ultrastructure by cryo soft X-ray tomography. J. Struct. Biol. 177, 179–192. Parkinson, D.Y., McDermott, G., Etkin, L.D., Le Gros, M.A. & Larabell, C.A. (2008) Quantitative 3-D imaging of eukaryotic cells using soft X-ray tomography. J. Struct. Biol. 162, 380–386. Pereiro, E., Nicolas, J., Ferrer, S. & Howells, M.R. (2009) A soft X-ray beamline for transmission X-ray microscopy at ALBA. J. Synchrotron Rad. 16, 505–512. Schneider, G., Guttmann, P., Rehbein, S., Werner, S. & Follath, R. (2012) Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging. J. Struct. Biol. 177, 212–223. Uchida, M., Sun, Y., McDermott, G., Knoechel, C., Le Gros, M.A., Parkinson, D., Drubin, D.G. & Larabell, C.A. (2011) Quantitative analysis of yeast internal architecture using soft X-ray tomography. Yeast 28, 227–236.
Supporting Information Additional Supporting information may be found in the online version of this article at the publisher’s website: Movie S1. Tomogram of Hek293 cell shown in Figure 3A. Movie scrolls through the virtual tomogram slices from the top of the cell to the bottom of the cell at 6 frames per second. Movie created with Quicktime Pro and compressed with H264 compression in Stomp (shineywhitebox.com). Scale bar – 2 μm. Movie S2. Tomogram of Hek293 cell shown in Figure 3B. Movie scrolls through the virtual tomogram slices from the top of the cell to the bottom of the cell at 6 frames per second. Movie created with Quicktime Pro and compressed with H264 compression in Stomp (shineywhitebox.com). Scale bar – 2 μm.
C 2014 The Authors C 2014 Royal Microscopical Society, 255, 65–70 Journal of Microscopy
doi: 10.1111/jmi.12139
Received 13 February 2014; accepted 22 April 2014
Biological applications of cryo-soft X-ray tomography E . D U K E ∗, K . D E N T ∗, M . R A Z I † & L . M . C O L L I N S O N ‡
∗ Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire, UK
†Secretory Pathways Laboratory, London Research Institute, Cancer Research UK, London, UK ‡Electron Microscopy Unit, London Research Institute, Cancer Research UK, London, UK
Key words. Cells, CLXM, Correlative, Cryo-soft X-ray tomography, Cryo-fluorescence, Cryo-preservation, Cryo-SXT, Organelles, Synchrotron.
Summary X-rays are used for imaging many different types of biological specimen, ranging from live organisms to the individual cells and proteins from which they are made. The level of detail achieved as a result of the imaging varies depending on both the sample and the technique used. One of the most recent technical developments in X-ray imaging is that of the soft X-ray microscope, designed to allow the internal structure of individual biological cells to be explored. With a field of view of 10–20 × 10–20 μm, a penetration depth of 10 μm and a resolution of 40 nm3 , the soft X-ray microscope neatly fits between the imaging capabilities of light and electron microscopes.
X-rays for imaging soft biological tissues Soft X-ray tomography (SXT) is similar in concept to electron tomography (Lucic et al., 2013). In SXT, the sample is tilted during image acquisition, and the resulting tilt series is reconstructed to give a stack of virtual images through the volume of the sample. The term ‘soft’ is used to differentiate the X-rays from ‘hard’ X-rays, which have a higher energy (>5–10 keV) and are able to penetrate into heavier materials such as bone and metals. Cells have very little contrast in hard X-ray microscopes, because low atomic number elements (carbon, oxygen, hydrogen, nitrogen) do not absorb high energy X-rays well. However, ‘soft’ X-rays with energy of 500 eV are preferentially absorbed by carbon rather than water (oxygen), and are able to penetrate through a depth of 10 μm of carbon/water. Given the nature of soft X-rays, a key aspect of most SXT microscopes is that the entire system is in vacuum. Thus, the cells must be embedded within a support matrix. Vitreous ice is the obvious choice, due to its low absorption of the X-rays. In addition, cryogenic temperatures reduce the ability of the X-rays to damage the samples during data collection. Thus, a unique Correspondence to: L. M. Collinson, Electron Microscopy Unit, London Research Institute, Cancer Research UK, London WC2A 3LY, UK. Tel: +44 (0) 207 269 3416; e-mail: [email protected]
C 2014 The Authors C 2014 Royal Microscopical Society Journal of Microscopy
property of the cryo-SXT microscope is that 3D ultrastructural information is obtainable from intact, unstained biological cells where the only sample preparation process has been cryo-preservation.
The cryo-soft X-ray microscope The cryo-SXT microscope is broadly similar to any other microscope, with certain parts tailored to the specific requirements of the X-rays used. The key to the success of the technique is the provision of a stable source of 500 eV X-rays. There are two alternatives for this – a synchrotron facility or a plasma source. The synchrotron has many advantages – it is intense so exposure times per image are short (1 s) and the energy of the X-rays can be varied, allowing compositional information to be collected via exploitation of elemental absorption edges. However, access to synchrotron facilities can be limited, and the time between proposal submission and actual beamtime (assuming successful application) can be long. The alternative, a nitrogen plasma source, has been found to work exceptionally well though data acquisition times are significantly longer than at the synchrotron (many hours compared with 10–30 min on the beamline). There are currently four cryo-SXT microscopes installed, operational and actively studying biological samples at synchrotrons worldwide, including the Advanced Light Source (ALS) in Berkeley (CA, USA) (Le Gros et al., 2005), BESSY II in Berlin (Germany) (Schneider et al., 2012) and ALBA in Barcelona (Spain) (Pereiro et al., 2009). Here, we focus on the newest of the microscopes, installed at the Diamond Light Source in Didcot (UK) (Fig. 1A) (Carzaniga et al., 2013). This cryo-SXT microscope (Xradia, now Carl Zeiss X-ray Microscopy Inc, Pleasanton, CA, USA) is currently operating with a nitrogen plasma source (Energetiq Technology Inc, Woburn, MA, USA), and is due to be connected to the synchrotron X-ray source in late 2014. The soft X-rays (from the synchrotron or plasma source) are focused onto the sample using a condenser (Fig. 1B). Once the X-rays have passed through the sample, they are then collected
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Fig. 1. Cryo-SXT microscope at the Diamond Light Source. (A) The cryo-SXT microscope (Xradia, now Carl Zeiss X-ray Microscopy Inc) is currently operating in off-line mode using a nitrogen plasma source (Energetiq). The microscope will move to a synchrotron X-ray source in 2014. (B) The main components inside the microscope vessel, including the capillary condenser (CC); sample cartridge containing a 3 mm grid mounted at the data collection position (SC); zone plate holder with the zone plate mounted at the tip (ZP); cryoshield (CS) and visible light microscope (VLM) objective.
and refocused onto the detector using the objective lens. For the soft X-ray microscope, the condenser is either a capillary lens or a zone plate and the objective is a zone plate. A zone plate is a series of concentric rings (zones) of alternating transparent and opaque material from which the X-rays diffract to form an image at the focal plane. It is the radius of the outermost ring of the zone plate that governs the theoretical resolution of the microscope. At present, the highest resolution zone plate in routine use for tomography of biological samples is 25 nm. The higher the resolution of the zone plate, the shorter its focal length. At 25 nm the zone plate must be placed 1–2 mm from the sample. Given that data collection requires sample rotation this can place a limit on the achievable angles, though this does vary depending on the type of sample mount used. In reality, the resolution of a data set is also limited by other factors including sample preparation, sample thickness and data reconstruction techniques. Preparing samples As with all imaging techniques, sample preparation is key to collecting high-quality data. The technique for sample preparation is dependent on the microscope chosen for data collection. In the current cryo-SXT facilities, samples are mounted using either capillaries or electron microscopes (EM) grids. Ultrathin-walled capillaries, used in the ALS microscope, are made by heating and pulling narrow-walled small diameter quartz capillary tubes such that their final internal diameter is approximately equal to the size of the sample being studied
(in the order of a few microns). The capillaries are then filled with a solution containing the samples in question and plunge frozen. This method lends itself particularly well to smaller cells that grow in suspension, such as bacteria and yeast. The BESSY II microscope currently requires bespoke EM grids designed specifically to allow sample rotation without colliding with the zone plate, so called ‘high tilt’ or ‘HZB-2’ grids, whereas the ALBA and Diamond microscopes can accept standard 3 mm transmission electron microscopy (TEM) grids. For both grid types, suspensions can be applied to the grid or adherent cells can be grown on grids that have been coated in a thin layer of holey carbon (Fig. 2A). It is important to have low confluency in order that individual cells can be viewed without overlap as the sample is tilted. Cells can be mapped prior to cryo-fixation using confocal microscopy, so that confluency can be assessed and cells expressing fluorophores located for correlative imaging (Fig. 2B). Plunge freezing in liquid nitrogen-cooled liquid ethane is currently the most popular method of cryo-preservation. This enables rapid vitrification and avoids the formation of hexagonal ice crystals that would otherwise disrupt cellular ultrastructure (Hagen et al., 2012). Immediately prior to freezing, fiducial markers are added to the grid to aid post-acquisition alignment of the tilt series (Fig. 2C). Grids are then blotted, another key step in obtaining high quality data (Fig. 2D). Over blotting before plunging can result in dehydration of cells and susceptibility to X-ray damage; under blotting leads to thick ice, resulting in poor penetration of the X-rays and weak contrast in the final image stack (particularly at high tilt angles). C 2014 The Authors C 2014 Royal Microscopical Society, 255, 65–70 Journal of Microscopy
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Fig. 2. Sample preparation workflow for cryo-SXT of whole mammalian cells. (A) Cells are grown on 3 mm finder grids (Diamond, ALBA) or HZB-2 grids (BESSY II). (B) Grids are screened for confluency and for cells expressing fluorophores in the confocal microscope for correlative experiments. (C) Gold fiducial markers are added to the grid to aid post-acquisition data alignment. (D) The grid is blotted to remove excess liquid, and (E) plunge frozen in liquid nitrogen-cooled liquid ethane. (F) An additional screening step can be included to map cells of interest and assess ice quality. (G) Frozen Hek293 cells with a fluorescent label highlighting mitochondria. (H) Grids are sent to the synchrotron beamline in a dry nitrogen shipper. Scale bars – B: 100 μm, G: 20 μm. C 2014 The Authors C 2014 Royal Microscopical Society, 255, 65–70 Journal of Microscopy
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The grids are plunge frozen (Fig. 2E), and transferred to cryostorage under liquid nitrogen. A cryo-fluorescence screening step can be used in the home laboratory (Fig. 2F) to locate cells of interest (Fig. 2G) and screen for artifacts. This step is critical for correlative imaging if the cryo-SXT microscope does not have an integrated fluorescence microscope (currently the case at ALBA), and can also help to maximise throughput at beamlines with integrated fluorescence microscopes (BESSY, Diamond). The grids are then shipped to the synchrotron for imaging (Fig. 2H). Imaging with a cryo-SXT microscope Cryo-SXT has benefited greatly from technical developments that have taken place in both EM and X-ray crystallography. Assuming a microscope that accepts samples grown on EM grids, then the mechanism for mounting the grids into the microscope takes much of its technology from cryo-TEM. Grids are mounted in holders and loaded into the microscope via a vacuum load-locked transfer device, which allows transfer of the samples into the microscope without needing to vent the microscope to atmospheric pressure. At beamlines equipped with an integrated visible light microscope with fluorescence capability, it is possible to further screen the grid after insertion into the cryo-SXT microscope to identify cells that are suitable for data collection. Once a cell has been selected, it is placed at the centre of sample rotation, which also coincides with the focal position of both the capillary condenser and zone plate objective lenses. Data acquisition proceeds via collection of a series of images at angular tilts, usually in the range of ±70°, a restriction imposed by the close proximity of the zone plate. At this point we term the data collected a tilt series. A strategy that takes into account the increased sample thickness at high angles when setting exposure times at each tilt has been found to be beneficial to final tomogram quality. Care must also be taken to ensure that the sample is not destroyed by the X-rays part-way through the data collection. At present, the procedure established to assess the damage during data collection is a comparison ‘by eye’ of the images taken at 0° before and after the tilt series acquisition. The images themselves are collected on a direct detection, back thinned CCD detector.
fiducial markers. The quality of this initial alignment has a huge influence on the quality of the sinogram that is calculated, and thus the feature resolution of the final tomogram. The reconstruction is then performed, usually with a back projection or iterative reconstruction algorithm. This leads to a stack of virtual images with high spatial resolution (25–40 nm3 ) through the volume of the cell. Contrast of cellular membranes in the tomograms resembles that of TEM, even though no heavy metal stains are added to the cells (Fig. 3). Indeed, contrast is directly related to the composition of the cellular structures, raising the possibility of automatic recognition of cellular structures by their density alone, measured as the linear absorption coefficient (LAC) (Parkinson et al., 2008). In the case where a complete rotation range can be collected (at the ALS using capillaries), it is possible to automatically segment organelles in the tomogram using the LAC. In the case of a limited tilt series (BESSY, ALBA, Diamond), the relationship of the LAC to cellular membranes is more complex, and automation of the segmentation process is less straightforward. Finding structures of interest using cryo-SXT A number of cellular structures can be recognised in soft Xray tomograms by their morphology alone, based on prior information from electron microscopy. The cell nucleus, nuclear envelope, plasma membrane, mitochondria and endoplasmic reticulum (ER) are easily discernible and can be followed through sequential images in the tomogram for 3D analysis (Fig. 3A,B and Movies S1,S2). Resolution is sufficient to see cristae in mitochondria, the lumen of the ER (Fig. 3C), the space between the two lipid bilayers of the nuclear envelope and nuclear pore complexes (Duke et al., 2013). An atlas of these and other organelles with less characteristic morphologies is gradually being compiled by the cryo-SXT community (Muller et al., 2012), including endosomes (Fig. 3D) identified using gold particles targeted to internalised receptors (Fig. 3E) (Duke et al., 2013). Features of interest may also be identified using fluorescent markers and a correlative workflow (Fig. 2), which has so far proved valuable in the study of mitochondria in yeast (Parkinson et al., 2008), virus-induced vesicular structures (Hagen et al., 2012) and autophagy (Duke et al., 2013) in mammalian cells.
Reconstructing data To date, cryo-SXT tilt series have been reconstructed into tomograms using a range of software packages, all originally developed for EM tomography. Therefore, at present, no account is taken of the properties of the X-rays (as opposed to electrons) or that the depth of focus of the X-rays is typically less than the sample thickness (for mammalian cells). Despite this, the results are quite spectacular. To create the tomogram from the tilt series, the background corrected images are first aligned to each other using the gold
The cryo-SXT niche A cryo-soft X-ray tomogram allows the scientist to build up a 3D image of the near-native state cell (Fig. 3F,G), which shows with stunning clarity the morphology, interactions and complexities of subcellular organelles. Volumetric analysis of cells and organelles is more accurate than light microscopy due to the resolution of the technique, and more accurate than electron microscopy due to the absence of shrinkage artifacts (Uchida et al., 2011). The absence of heavy metal C 2014 The Authors C 2014 Royal Microscopical Society, 255, 65–70 Journal of Microscopy
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Fig. 3. Ultrastructure of whole, unstained Hek293 cells imaged by cryo-SXT at the Diamond Light Source. (A)–(D) Virtual slices from tomograms collected using the cryo-SXT microscope at the Diamond Light Source, currently operating with a nitrogen plasma source. Tilt series were collected from ±60° and the resulting images were reconstructed into a tomogram using the SIRT algorithm in IMOD (http://bio3d.colorado.edu/imod). (A) Three image planes from a tomoX stack, moving from the bottom (left panel) to the top (right panel) of the cell. At the bottom of the cell (left panel), the holes in the QuantifoilTM carbon support are visible (dashed circles) as well as the 250 nm gold fiducial markers (arrowheads). Endoplasmic reticulum (ER) and mitochondrial networks (m) with fine connections (arrows) can be followed through the stack. See also Movie S1. (B) As in (A), but also showing the plasma membrane (PM). See also Movie S2. (C)–(E) Ultrastructure is similar to that seen in TEM, even though no heavy metal stains are used. (C) Cristae can be seen inside mitochondria (m) and the lumen of the endoplasmic reticulum (ER) is visible. (D) Endosomes with tubular extensions are visible, identified by comparison with, (E) Transferrin-receptor-gold loaded endosomes imaged by cryo-SXT at the U41-TXM microscope at BESSYII in Berlin (Duke et al., 2013). (F) and (G) XYZ planes showing the sample thickness and three dimensional nature of the data from the tomogram in panel A (F) and B (G) (screenshots from IMOD). Scale bars – 2 μm. C 2014 The Authors C 2014 Royal Microscopical Society, 255, 65–70 Journal of Microscopy
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stains allows detection of compositional information, used to great effect in analysis of iron-containing red blood cells infected with the malaria parasite P. falciparum (Hanssen et al., 2012). Fine connections can be seen between individual mitochondria that are not easily visible by either light or electron microscopy (Fig. 3) (Duke et al., 2013) enabling studies of mitochondrial networks in health and disease states. Volatile structures that are prone to chemical fixation artifacts, such as tubular endosomes and autophagosomes, are optimally captured by cryo-preservation and can be analysed in 3D through the image stack (Duke et al., 2013), with wider applications to studies of organelle biogenesis and membrane trafficking. Though cryo-SXT may currently lack the resolution to place macromolecular structures into cellular context, there is already a proven niche for cryo-SXT in the analysis of the near native-state cell. It is still early days in the development of the cryo-SXT field, with technological advances in zone plates, correlative workflows and tailored software likely to drive forward the scope of scientific applications. Indeed, there has been a shift in publications from development of the technology and proof-of-principle studies to incorporation as an imaging tool in bioscience investigations. With increasing uptake from scientists in collaboration with imaging experts, it would be unwise to underestimate the potential of this fledgling imaging technique.
Acknowledgements We gratefully acknowledge funding from Cancer Research UK (MR & LC). The research leading to the results shown in Figure 3(E) received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement N°283570). ED and KCD would like to thank the Xradia team for their outstanding dedication to the development of the soft X-ray microscope and to Energetiq for their support in the implementation of the plasma source. We would also like to thank Dr Raffaella Carzaniga, Dr Marie-Charlotte Domart and Elzbieta Kubiak-Goswami for the photographs in Figure 2.
References Carzaniga, R., Domart, M.C., Collinson, L. M. & Duke, E. (2013) Cryo-soft X-ray tomography: a journey into the world of the native-state cell. Protoplasma 251, 449–458. Duke, E.M.H., Razi, M., Weston, A., et al. (2013) Imaging endosomes and autophagosomes in whole mammalian cells using correla-
tive cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM). Ultramicroscopy. Epub ahead of print. doi: 10.1016/j.ultramic. 2013.10.006 Hagen, C., Guttmann, P., Klupp, B., Werner, S., Rehbein, S., Mettenleiter, T.C., Schneider, G. & Grunewald, K. (2012) Correlative VISfluorescence and soft X-ray cryo-microscopy/tomography of adherent cells. J. Struct. Biol. 177, 193–201. Hanssen, E., Knoechel, C., Dearnley, M., Dixon, M.W., Le Gros, M., Larabell, C. & Tilley, L. (2012) Soft X-ray microscopy analysis of cell volume and hemoglobin content in erythrocytes infected with asexual and sexual stages of Plasmodium falciparum. J. Struct. Biol. 177, 224–232. Le Gros, M.A., McDermott, G. & Larabell, C.A. (2005) X-ray tomography of whole cells. Curr. Opin. Struct. Biol. 15, 593–600. Lucic, V., Rigort, A. & Baumeister, W. (2013) Cryo-electron tomography: the challenge of doing structural biology in situ. J. Cell. Biol. 202, 407– 419. Muller, W.G., Heymann, J.B., Nagashima, K., Guttmann, P., Werner, S., Rehbein, S., Schneider, G. & McNally, J.G. (2012) Towards an atlas of mammalian cell ultrastructure by cryo soft X-ray tomography. J. Struct. Biol. 177, 179–192. Parkinson, D.Y., McDermott, G., Etkin, L.D., Le Gros, M.A. & Larabell, C.A. (2008) Quantitative 3-D imaging of eukaryotic cells using soft X-ray tomography. J. Struct. Biol. 162, 380–386. Pereiro, E., Nicolas, J., Ferrer, S. & Howells, M.R. (2009) A soft X-ray beamline for transmission X-ray microscopy at ALBA. J. Synchrotron Rad. 16, 505–512. Schneider, G., Guttmann, P., Rehbein, S., Werner, S. & Follath, R. (2012) Cryo X-ray microscope with flat sample geometry for correlative fluorescence and nanoscale tomographic imaging. J. Struct. Biol. 177, 212–223. Uchida, M., Sun, Y., McDermott, G., Knoechel, C., Le Gros, M.A., Parkinson, D., Drubin, D.G. & Larabell, C.A. (2011) Quantitative analysis of yeast internal architecture using soft X-ray tomography. Yeast 28, 227–236.
Supporting Information Additional Supporting information may be found in the online version of this article at the publisher’s website: Movie S1. Tomogram of Hek293 cell shown in Figure 3A. Movie scrolls through the virtual tomogram slices from the top of the cell to the bottom of the cell at 6 frames per second. Movie created with Quicktime Pro and compressed with H264 compression in Stomp (shineywhitebox.com). Scale bar – 2 μm. Movie S2. Tomogram of Hek293 cell shown in Figure 3B. Movie scrolls through the virtual tomogram slices from the top of the cell to the bottom of the cell at 6 frames per second. Movie created with Quicktime Pro and compressed with H264 compression in Stomp (shineywhitebox.com). Scale bar – 2 μm.
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