Imaging cells and sub-cellular structures with ultrahigh resolution full-field X-ray microscopy C.C. Chien a, b , P.Y. Tseng a , H.H. Chen a , T.E. Hua a , S.T. Chen a , Y.Y. Chen a , W.H. Leng a , C.H. Wang a , Y. Hwu a, b, c, d, ⁎, G.C. Yin e , K.S. Liang f , F.R. Chen b , Y.S. Chu g , H.I. Yeh h , Y.C. Yang h , C.S. Yang i , G.L. Zhang j , J.H. Je k , G. Margaritondo l a Institute of Physics, Academia Sinica, Taipei 115, Taiwan b Engineering and System Science, National Tsing Hua University, Hsinchu 300, Taiwan c Institute of Optoelectronic Sciences, National Taiwan Ocean University, Keelung 202, Taiwan d Advanced Optoelectronic Technology Center, National Cheng Kung University, Tainan 701, Taiwan e National Synchrotron Radiation Research Center, Hsinchu 300, Taiwan f Electrophysics Department, National Chiao Tung University, Hsinchu 300, Taiwan g NSLS-II, Brookhaven National Laboratory, Upton, NY 11973‐5000, USA h Mackay Memorial Hospital, Taipei 104, Taiwan i Center for Nanomedicine, National Health Research Institutes, Miaoli 350, Taiwan j Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China k X-ray Imaging Center, Pohang University of Science and Technology, Pohang 790‐784, Republic of Korea l Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland abstract article info Available online 21 April 2012 Keywords: X-ray microscopy Phase contrast radiology Fresnel phase zone plate Subcellular organelle Our experimental results demonstrate that full-field hard-X-ray microscopy is finally able to investigate the internal structure of cells in tissues. This result was made possible by three main factors: the use of a coherent (synchrotron) source of X-rays, the exploitation of contrast mechanisms based on the real part of the refrac- tive index and the magnification provided by high-resolution Fresnel zone-plate objectives. We specifically obtained high-quality microradiographs of human and mouse cells with 29 nm Rayleigh spatial resolution and verified that tomographic reconstruction could be implemented with a final resolution level suitable for subcellular features. We also demonstrated that a phase retrieval method based on a wave propagation algorithm could yield good subcellular images starting from a series of defocused microradiographs. The con- cluding discussion compares cellular and subcellular hard-X-ray microradiology with other techniques and evaluates its potential impact on biomedical research. © 2012 Elsevier Inc. All rights reserved. 1. Introduction The development of new microscopy methods is a key element in the historical progress of biological and biomedical research. A land- mark for each microscopy technique is the detection of individual cells and of their internal structure (Fischer et al., 2011; Marsh et al., 1971; Marton, 1941; Porter et al., 1945; Tanaka and Fukudome, 1991). For over one century, X-rays did play a fundamen- tal role in biomedical research — but they were so far unable to image internal cell features in tissues. In a series of recent experiments, we achieved this important objective. Our strategy was based on a combination of factors to improve the image contrast and spatial resolution. One of them was the use of highly bright and coherent X-rays emitted by synchrotron sources. The coher- ence specifically facilitated the task of focusing the radiation to improve the resolution. Furthermore, it enabled us to improve the image con- trast by exploiting mechanisms (Cloetens et al., 2002; Hwu et al., 2002) based on the real part of the refractive index rather than on ab- sorption (the imaginary part). These mechanisms – conventionally called “phase contrast radiology”– strongly enhance the contrast be- tween soft tissues in biological specimens. Our experiments used short-wavelength (~1 Å) X-rays capable to penetrate thick specimens in a natural state. On the contrary, pre- vious microradiology tests were based on long-wavelength soft-X- rays in the “water window” (23–45 Å) that maximize the contrast between carbon-containing areas and water (Jacobsen et al., 2002; Kirz et al., 1994). With advanced soft-X-ray optics, subcellular struc- tures could be observed in hydrated states (Larabell and Nugent, 2010; McDermott et al., 2009). However, soft-X-rays cannot pene- trate specimens thicker than a few cells (≈10 μm) and are thus un- suitable for cell studies at the tissue level. This is a rather severe Biotechnology Advances 31 (2013) 375–386 ⁎ Corresponding author at: Institute of Physics, Academia Sinica, Taipei 115, Taiwan. Fax: +886 2 2789 6721. E-mail address: phhwu@sinica.edu.tw (Y. Hwu). 0734-9750/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2012.04.005 Contents lists available at SciVerse ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv