Imaging Fully Hydrated Whole Cells by Coherent X-Ray Diffraction Microscopy Daewoong Nam, 1,2 Jaehyun Park, 1 Marcus Gallagher-Jones, 1,3 Sangsoo Kim, 1 Sunam Kim, 1 Yoshiki Kohmura, 1 Hisashi Naitow, 1 Naoki Kunishima, 1 Takashi Yoshida, 4 Tetsuya Ishikawa, 1 and Changyong Song 1, * 1 RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan 2 Department of Physics, Pohang University of Science and Technology, Pohang 790-784, Korea 3 Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, United Kingdom 4 Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan (Received 19 May 2012; published 28 February 2013) Nanoscale imaging of biological specimens in their native condition is of long-standing interest, in particular with direct, high resolution views of internal structures of intact specimens, though as yet progress has been limited. Here we introduce wet coherent x-ray diffraction microscopy capable of imaging fully hydrated and unstained biological specimens. Whole cell morphologies and internal structures better than 25 nm can be clearly visualized without contrast degradation. DOI: 10.1103/PhysRevLett.110.098103 PACS numbers: 87.64.Bx, 42.30.Rx, 87.59.e The invention of compound microscopes, pioneered by A. v. Leeuwenhoek in the late 17th century, and subsequent developments to investigate biological specimens have drastically expanded our understanding of microsystems by providing views into structural details far beyond those perceivable by the human eye. The tremendous interest in unveiling ultrastructures by introducing new imaging methodologies with improved resolution and functions has not dwindled since then [1]. Progress toward high- resolution microscopy has not only deepened our understanding, but also invoked keen interest in imaging specimens at a condition closer to their physiological state. Cryogenic preservation of biological specimens has been adopted widely as a good proximity to preserving the native state. It, however, is accompanied by stringent sample treatments that can lead to unwanted structural alterations [2]. Realizing that most biological systems exist in a hydrated environment, imaging specimens under full hydration is undoubtedly ideal. Optical microscopy has routinely imaged biological specimens in solution, but image resolution usually remains on a cellular scale. Superresolution techniques have improved this to better than 100 nm, but elaborate sample handling is necessary [3–6]. Moreover, acquired images often visualize only local portions of a whole specimen where fluorescent dyes are located. Developments in electron microscopy to observe whole, hydrated cells with a nanoscale image resolution have been noteworthy [7,8]. Restrictions on sample conditions and degraded image contrast in solution, however, remain a major obstacle for general applications in bioimaging. The shorter wavelength of x rays holds the promise of higher resolution, introducing various imaging modalities [9]. Differential attenuation of x rays in specimens allows absorption contrast microscopy with either lens or contact registry [10]. Techniques contingent on the phase contrast of specimens such as Zernike phase contrast, holography, Talbot interferometry, etc., have been actively developed, showing good contrast even for low Z materials [11]. Here we introduce a newly developed wet coherent diffraction imaging (wet-CDI) technique capable of imag- ing fully hydrated, unstained, whole cells. The CDI tech- nique has inherent advantages essential for imaging specimens in solution. Images are acquired by inverting the coherent diffraction patterns recorded finer than the Nyquist sampling frequency with the phases retrieved via numerical iterations [12]. As the diffraction patterns are invariant for any translational sample drift under a plane wave incidence, CDI provides the ultimate imaging stabil- ity, critical for high resolution in solution. The straightfor- ward setting in CDI, with no requirement for lens or extrinsic sample treatment, has rapidly expanded applica- tions to biological specimens and nanostructured materials [12–14]. Excellent image contrast is noteworthy in CDI as demonstrated by imaging single, unstained viral particles [15,16]. The image contrast in CDI is not limited by the absorption, but being further enhanced by the phase dif- ference of x rays while interacting with specimens. This facilitates robust high contrast imaging for a wide range of x-ray energies and better addresses its advantage in imag- ing hydrated specimens compared with lens based electron or x-ray microscopy [8,9,17–19]. To establish the wet-CDI technique, we have developed two key components: a hydrated specimen holder and a helium ambiance x-ray diffraction microscope. The sche- matics of the diffraction microscope are shown in Fig. 1. The hydrated specimen holder is made of two Si 3 N 4 mem- branes with 5  5 arrays of 30 nm thin windows (Silson Ltd.). Cells were dispersed onto the windows along with a buffer solution, and were sealed up using another mem- brane, preserving the specimens in a fully hydrated PRL 110, 098103 (2013) PHYSICAL REVIEW LETTERS week ending 1 MARCH 2013 0031-9007= 13=110(9)=098103(5) 098103-1 Ó 2013 American Physical Society