International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 9.3, pp. 235-237

Section 9.3.3. High-resolution imaging of yeast

D. Shapiroa*

aAdvanced Light Source, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd, MS 2–400, Berkeley, CA 94720, USA
Correspondence e-mail: dashapiro@lbl.gov

9.3.3. High-resolution imaging of yeast

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The X-ray dose required to image a given volume of protein is nearly independent of energy above the oxygen K edge. At the same time, the photon flux required to image the same volume increases with E2 because of the energy dependence of the scattering cross section (Howells et al., 2009[link]). For this reason, it is advantageous to use the lowest energy commensurate with the desired resolution of 5–10 nm. Commercially available charge-coupled device (CCD) detectors can easily provide a scattering angle of 0.1 radians [a 1 inch detector placed 5 inches from the sample (1 inch = 2.54 cm)], which results in a half-period resolution of 8 nm when using 750 eV X-rays. Furthermore, a cell with a diameter of 3 µm would have an oversampling ratio (number of intensity samples per speckle) of at least ten in this geometry if the detector has 20 µm pixels.

In this particular case, a Princeton Instruments CCD (PI-MTE:1300) is placed 136 mm downstream of a freeze-dried yeast cell using the CXDM instrument on Beamline 9.0.1 of the Advanced Light Source (ALS). The yeast cell is illuminated by a coherent beam of 750 eV X-rays defined by a 5 µm pinhole located 25 mm upstream. The incident intensity of 4 × 106 photons s−1 µm−2 is high enough to cause rapid structural changes to the cell (discussed in the next section), so the sample requires pre-irradiation for about 30 minutes prior to collection of the final data set intended for reconstruction. The final data set, shown in Fig. 9.3.3.1[link], is a 1024 × 1024 pixel subset of the full CCD and extends to a resolution of 11 nm at the edge and 7.8 nm in the corner. Speckles extend to the corner of the data set after a total exposure of 406 s and to the edge after 226 s.

[Figure 9.3.3.1]

Figure 9.3.3.1 | top | pdf |

Diffraction pattern (left) and reconstruction (right) of a freeze-dried budding yeast cell. The diffraction pattern, measured on Beamline 9.0.1 of the ALS, extends to a half-period resolution of 11 nm and required 226 s of X-ray exposure. The blue regions of the diffraction pattern represent zeroes (noisy measurements or pixels lost behind the beamstop) and were left unconstrained during phase retrieval. The image represents the complex-valued X-ray wavefield after passing completely through the scattering potential, but propagated to the interior plane with the smallest support. The X-ray phase is represented as image hue and magnitude as brightness. The PRTF (not shown) never dips below 0.5, indicating that the magnitudes were adequately phased to the corner of the recorded data. The large scale bar is 1 µm, while the inset scale bar is 100 nm. The reconstruction presented here is the average of 25 independent reconstructions, each starting with a different set of random phases. Each reconstruction required 2000 iterations of the hybrid input–output algorithm and took about 14 s to complete on an nVidia Tesla C1060 graphics processing unit.

The full reconstruction of this type of data set can only proceed once a high-fidelity support is determined. Algorithmic support determination using Shrinkwrap, a variation on the hybrid input–output algorithm, is straightforward for objects with sharp boundaries, as is often the case in the material sciences (Marchesini et al., 2003[link]; Chapman et al., 2006[link]). However, biological samples which often have soft boundaries require manual intervention in the early stages of reconstruction. The soft-edge problem is exacerbated by the loss of low spatial frequency information behind the beamstop. A combination of Shrinkwrap support adjustments and intuitively reasonable manual adjustments are made until the algorithm is stable to automatic adjustment. This point is found when further Shrinkwrap adjustments no longer alter the shape of the support but just its tightness. High-fidelity reconstructions of complex-valued objects are not possible with a loose support (Fienup, 1987[link]). Once the support is found, the final image is produced as the average over many reconstructions, all using the same support, which are started from different random phase sets. This averaging procedure, discussed earlier, reduces features that are primarily due to noise and provides a measure of the reproducibility of the recovered phases and therefore an estimate of the resolution. The final reconstruction shown in Fig. 9.3.3.1[link] had PRTF values above 0.5 for spatial frequencies extending to the edge of the data set or 11 nm resolution. The inset image clearly shows features of the order of 15 nm in size. In general, it is not possible to identify cell organelles without labelling specific proteins. However, a correlative study of yeast which combines high-resolution CXDM with optical fluorescence would be a powerful tool for the cell-biology community. This technique has already been demonstrated at lower resolutions using a transmission X-ray microscope (Le Gros et al., 2009[link]).

9.3.3.1. Radiation damage

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High-resolution imaging of single particles with X-rays requires a large radiation dose because of the very strong dependence of the scattering cross section on spatial frequency and because, in the single-particle case, there is none of the coherent amplification one obtains when many identical copies of the particle are arranged into a crystal. This large dose means that either the sample must be protected from morphological changes induced by radiation exposure, most effectively through cryogenic techniques, or that the obtained images will represent an altered form of the original sample. Cryoprotection of hydrated cells has been successfully used in electron microscopy and lens-based X-ray microscopy for some time, but the sample preparation requirements of diffractive imaging are more severe, so the development of cryotechniques has been slower. The requirements of a finite sample support and that the sample be maintained on a zero-scattering background are extraordinarily difficult to achieve when that background consists of a micron-thick layer of ice. This is an active area of research by several X-ray diffraction microscopy groups.

For the case of dry cells, various preparation techniques (freeze drying or chemical fixation and dehydration) can preserve the large-scale internal structure, such as the size and shape of large organelles, but the ultrastructure will inevitably show artifacts of the drying process. Furthermore, exposure to ionizing radiation results in the well known shrinkage problem. Studies with a transmission X-ray microscope indicate that X-ray induced shrinkage primarily produces a higher-density but smaller version of the original cell (Jearanaikoon & Abraham-Peskir, 2005[link]). The effect of cell shrinkage on the X-ray diffraction pattern is shown in Fig. 9.3.3.2[link]. A freeze-dried cell is repeatedly exposed to 750 eV X-rays from Beamline 9.0.1 of the ALS. Each exposure is 30 s and delivers an X-ray dose of approximately 5 × 108 Gy. The sample is stable for the first two exposures but then experiences a rapid collapse, followed by a slow but continuous shrinkage. The collapse is apparent from the elongated speckles, which indicate a sample for which the diameter is changing during the exposure while its relative structure is maintained. Overall, the sample loses about 25% of its volume prior to the final exposure used for reconstruction (Shapiro et al., 2005[link]; Thibault et al., 2006[link]). Over the course of this exposure series the total scattered signal does not change, indicating that the total mass of the sample remains intact. The rapid change in the diffraction pattern during the early exposures means that successful imaging experiments require pre-irradiation of the sample. The slow but continuous shrinkage of dry samples with further dose means that the resolution of the three-dimensional images will necessarily be reduced. Indeed, Nishino et al. (2009[link]) observed reduced resolution in their three-dimensional reconstruction of a dry chromosome because of morphological changes which occurred during data collection. Hence, full three-dimensional imaging of cells by diffractive methods requires cryogenic protection against radiation damage. Predictions based on a calculation of the cross section for coherent scattering by a smooth dielectric indicate that 10 nm resolution imaging of frozen hydrated organic matter should be possible using soft X-rays at currently available synchrotron sources (Howells et al., 2009[link]). This limit is arrived at through a comparison of the radiation dose required for imaging and the dose at which radiation damage has been empirically observed at different length scales. However, it seems plausible that the presence of many identical particles within a cell could be exploited to provide super-resolution information.

[Figure 9.3.3.2]

Figure 9.3.3.2 | top | pdf |

Exposure to ionizing radiation causes shrinkage of organic matter. Each image in this series is a section of a measured diffraction pattern from a freeze-dried yeast cell. The images were taken sequentially and each represents an additional X-ray dose of 5 × 108 Gy. After a cumulative dose of 1 × 109 Gy, the cell undergoes a rapid collapse [apparent from the elongated speckles in images (3)–(5)] followed by continued shrinkage at a reduced rate. The X-rays used had an energy of 750 eV and a dose of 5 × 108 Gy was adequate for reconstruction at 30 nm resolution. (Reproduced from Shapiro, 2004[link]).

9.3.3.2. Low-dose three-dimensional imaging; low damage potential of stereoscopic viewing

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Diffraction imaging in three dimensions proceeds as it does in standard X-ray tomography. That is, two-dimensional data are recorded from many angular orientations of the sample and then assembled into a three-dimensional data set. In this case, however, the data are recorded in reciprocal space and the individual data sets only need to be registered with respect to the angular coordinate due to the Fourier shift theorem. In the absence of any additional information, the angular sampling of reciprocal space is determined by the Crowther resolution,[k_{C}={{1}\over{\Delta \theta D}},]where D is the object diameter and Δθ is the angular separation of the two-dimensional data sets. This is the spatial frequency at which the unmeasured Fourier components, those in between the measured Ewald sphere segments, can be properly interpolated from the measured data. Diffraction microscopy, however, requires the addition of information in the form of real-space constraints. This additional information allows for the calculation of not only the missing reciprocal-space phases but also a limited number of missing magnitudes. Chapman et al. (2006[link]) showed that Δθ could in fact be up to four times larger than required by the Crowther relation, with kC matching the numerical aperture of the imaging system. Thus, three-dimensional reconstructions could take place with nearly isotropic diffraction-limited resolution with only about 150 angular orientations of the sample.

Stereoscopic viewing can provide a significant degree of three-dimensional perception of an extended object while only increasing the total radiation exposure by a factor of two. In principle, according to the dose-fractionation theorem of Hegerl and Hoppe, full three-dimensional visualization of a given resolution element should not require a dose any higher than two-dimensional visualization of the same element with the same statistical accuracy (Hegerl & Hoppe, 1976[link]). This theorem provides hope that high-resolution imaging in three-dimensions, perhaps even of dry specimens, is possible, but in practice this is very difficult to achieve and low-dose imaging techniques are only now being explored by the CXDM community. Stereoscopic viewing should be considered the preliminary low-dose technique of choice. One particular advantage is the rapid reconstruction (compared with full three-dimensional reconstructions) which makes possible in situ sample inspection. Fig. 9.3.3.3[link] shows a stereo image of a chemically dried budding yeast cell. When viewed stereoscopically, with the viewer's focus in front of the image, the three-dimensional arrangement of a group of vesicles in the mother cell can be visualized.

[Figure 9.3.3.3]

Figure 9.3.3.3 | top | pdf |

Stereo image of a budding yeast cell. This budding yeast cell was chemically fixed with gluteraldehyde and dehydrated in acetone. The images have an angular separation of 10° and a pixel size of 11 nm. The three-dimensional arrangement of a group of small vesicles in the mother cell can be visualized when viewed stereoscopically (with the viewer's focus in front of the image). The scale bar is 500 nm.

References

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