78 79 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Studying biological structures using electron ptychography Related publication: Zhou L., Song J., Kim J. S., Pei X., Huang C., Boyce M., Mendonça L., Clare D., Siebert A., Allen C. S., Liberti E., Stuart D., Pan X., Nellist P. D., Zhang P., Kirkland A. I. &Wang P. Low-dose phase retrieval of biological specimens using cryo-electron ptychography. Nat. Commun. 11 , 2773 (2020). DOI: 10.1038/s41467-020-16391-6 Publication keywords: Cryo-electron microscopy; Electron ptychography; Rotavirus; HIV-1 virus like particles C ryo-electron microscopy (Cryo-EM) is an essential tool for obtaining high-resolution structural studies of biological systems. However, unstained biological samples are extremely sensitive to radiation and the images obtained have low contrast and low signal-to-noise ratios. There are techniques to overcome these limitations. However, it is more challenging to obtain high-resolution images for heterogeneous samples, specimens at low concentration, low symmetry structures and small or flexiblemolecules. An international teamof researchersworkingat Diamond’s ElectronPhysical Sciences ImagingCentre (ePSIC) set out todevelopanewmethod for imaging biological structures using electrons. Ptychography is an alternative method based on scanning diffraction microscopy. It uses a probe to illuminate the sample and records a series of far-fielddiffractionpatterns.The teamdemonstrated that this novelmethod is suitable for studies ofmany biologically important structures, including small molecules belowa size that can be imaged using conventional Cryo-EM. Determining the three-dimensional (3D) structures of biological macromolecules and assemblies at high resolution in their native states has driven significant and sustained efforts to develop electron microscopy (EM) techniques, most notably phase contrast cryo-EM 1 . However, unstained biological samples embedded in thin vitreous ice are essentially pure phase objects that are extremely radiation sensitive 2 . As a result cryo-EM images must be recorded at very low electron dose and have a low signal-to-noise ratio and low contrast. In conventional cryo-EM high defocus values are used to boost low spatial frequencies, improving contrast, but at the expense of corrupting informationat intermediateandhighspatialfrequenciesduetorapidoscillations in the phase contrast transfer function. Despite these fundamental limitations, classaveraging largenumbersofhomogeneousobjectsandusingdirectelectron detectors to improve the signal-to-noise ratio has enabled a large number of 3D biological structures at close to atomic resolution to be solved 3 .There is therefore a need to develop new methods that improve information transfer over a wide range of spatial frequencies, which work efficiently at low electron dose and which can be used over wide fields of view. Ptychography is one suchmethod based on scanning diffractionmicroscopy, as originally proposed by Hoppe 4 . This approach uses a probe to illuminate the sample and records a series of far-field diffraction patterns as a function of probe position, as illustrated in Fig. 1, to recover the sample exit plane wavefunction using one of several iterative or direct methods. Ptychography has been most widely with X-rays as the wavefunction recovered can exceed the spatial resolution that can be obtained using conventional optics. Due to its efficient phase recovery, robustness under low electron dose conditions 5 and the quantitative recovery of the 3D wavefunction, electron ptychography has significant potential for application in the structure determination of biological samples. An important advantage of this method is the ability to tune information transfer to maximise low or high spatial frequencies by altering the probe convergence angle. In the work reported in Nature Communications, a team from ePSIC, the Electron Bio-Imaging Centre (eBIC) and Oxford and Nanjing Universities, demonstrate the first application of cryo-electron ptychography (cryo-EPty) using a defocused electron probe to image biological structures. Electron ptychographic datasets were recorded 300kV using a double aberration corrected JEOL ARM300CF at ePSIC on a 256 × 256 pixel Merlin Medipix3 direct electron detector. The phase and amplitude of the complex specimen exit wavefunction were recovered using the ptychographic iterative algorithm (ePIE). To be generally applicable to structural studies of biological materials in a cryo state, ptychographic reconstruction must be effective under low dose conditions, similar to those used for conventional EM imaging. Defocused probe ptychography uses a relatively large probe (25-30 nm in diameter for the experiments reported here) and hence it is optimised to scan a large sample area with a small number of probe positions, reducing the total dose if the overlap between neighbouring probe positions is optimised. Fig. 2 shows ptychographic phase reconstructions of rotavirus double-layered particles (DLPs) embedded in vitrified ice for doses between 22.8 e/Å 2 and 5.7 e/Å 2 . The ptychographic phase of rotavirus DLPs shows strong contrast from the virus particles, where both the capsid trimers of viral protein 6 (VP6) and the channels between these can be clearly seen. The observation of these features and the resolution of the viral capsid’s symmetric elements are consistent with those observed using conventional defocused transmission electron microscope (TEM) images and with the known 3D structure (PDB ID 3KZ4). The final requirement for the application of ptychographic reconstruction in structural and cellular biology is the ability to recover information from large fields of view corresponding to cellular ultrastructure. To demonstrate this, a micrometre (1.14 × 1.14 μm) area of a resin-embedded Adenovirus- infected cell was reconstructed at a dose of 27 e/Å 2 (Fig. 3). The ptychographic phase shows good visibility of ultrastructural features of varied size, including cytoskeletal elements, viral particles, vacant vesicles, transport vesicles and free ribosomes. Moreover, as already noted, reconstruction of the ptychographic phase preserves low spatial frequency information, facilitating the location of these key molecular features in a cellular context. In conclusion, the studies reported demonstrate that cryo-electron iterative ptychography provides high contrast quantitative phase recovery at an electron dose of 5.7 e/Å 2 over wide fields of view making this approach suitable for biological macromolecular imaging.This approach provides tunable, continuous wide-band information transfer including low spatial frequencies that are inaccessible using conventional phase contrast imaging. In addition, the efficiency of the phase recovery using ptychography provides higher signal-to- noise data than phase contrast imaging in cryo-EM, which potentially reduces the particle numbers required for 3D reconstruction, facilitating 3D classification of heterogeneous specimens at low concentration or from structures with low symmetry. References: 1. Adrian M. et al. Cryo-electron microscopy of viruses. Nature 308 , 32–36 (1984). DOI: 10.1038/308032a0 2. Henderson R.The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules. Q. Rev. Biophys. 28 , 171–193 (1995). DOI: DOI: 10.1017/S003358350000305X 3. Bartesaghi A. et al. 2.2 å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor. Science. 348 , 1147–1151 (2015). DOI: 10.1126/science.aab1576 4. Hoppe,W. Diffraction in inhomogeneous primary wave fields 1. Principle of phase determination from electron diffraction interference. Acta Crystallogr. A 25, 495-501 (1969). 5. Song J. et al. Atomic Resolution Defocused Electron Ptychography at Low Dose with a Fast, Direct Electron Detector. Sci. Rep. 9 , 3919 (2019). DOI: 10.1038/s41598-019-40413-z Funding acknowledgement: National Natural Science Foundation of China (11874199); UK Medical Research Council (MR/N00065X/1).We thank Diamond Light Source for access and support in the use of the electron Physical Science Imaging Centre (Instrument E02, EM17918) that contributed to the results presented. Corresponding author: Prof. Angus Kirkland, University of Oxford,
[email protected] Imaging andMicroscopy Group ePSIC Figure 1: Schematic diagram of the optical configuration for cryo-electron ptychography. Figure 2: Reconstructed phase of rotavirus DLPs at doses of (a) 22.8 e/Å 2 ; (b) 11.3 e/Å 2 ; (c) 5.7 e/Å 2 . The red arrow in (c) indicates the VP6 trimers. Scale bars: 100 nm. Figure 3: (a) Micrometre scale reconstructed phase of an Adenovirus-infected cell recorded at a dose of 27 e/Å 2 ; (b) Magnified views of a viral particle; (c) A vacant vesicle; (d) A transport vesicle; and (e) Free ribosomes taken from regions indicated with orange squares in (a). Scale bars are (a) 300 nm and (b to e) 50nm.