Three-Dimensional X-ray Observation of Atmospheric Biological Samples by Linear-Array Scanning-Electron Generation X-ray Microscope System Toshihiko Ogura* Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Umezono, Tsukuba, Ibaraki, Japan Abstract Recently, we developed a soft X-ray microscope called the scanning-electron generation X-ray microscope (SGXM), which consists of a simple X-ray detection system that detects X-rays emitted from the interaction between a scanning electron beam (EB) and the thin film of the sample mount. We present herein a three-dimensional (3D) X-ray detection system that is based on the SGXM technology and designed for studying atmospheric biological samples. This 3D X-ray detection system contains a linear X-ray photodiode (PD) array. The specimens are placed under a CuZn-coated Si3N4 thin film, which is attached to an atmospheric sample holder. Multiple tilt X-ray images of the samples are detected simultaneously by the linear array of X-ray PDs, and the 3D structure is calculated by a new 3D reconstruction method that uses a simulated- annealing algorithm. The resulting 3D models clearly reveal the inner structure of the bacterium. In addition, the proposed method can easily be used for diverse samples in a broad range of scientific fields. Citation: Ogura T (2011) Three-Dimensional X-ray Observation of Atmospheric Biological Samples by Linear-Array Scanning-Electron Generation X-ray Microscope System. PLoS ONE 6(6): e21516. doi:10.1371/journal.pone.0021516 Editor: Akos Vertes, The George Washington University, United States of America Received February 4, 2011; Accepted May 30, 2011; Published June 23, 2011 Copyright: 2011 Toshihiko Ogura. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by KAKENHI Grant-in-Aid for Scientific Research (B) (No. 20300107) of the Japan Society for the Promotion of Science, and the Canon foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: t-ogura@aist.go.jp Introduction Imaging with nanometre-scale resolution is indispensable for many scientific fields such as biology, physics, chemistry, materials science and nanotechnology [1–5], and soft-X-ray microscopy is an important technique for producing such high-resolution images of unstained samples that are under atmospheric pressure or in water [6–9]. When combined with a diffractive zone plate and a synchrotron light source, the spatial resolution of soft-X-ray microscopy is approximately 10 nm [10,11], which is much higher than the resolution available with an optical microscope. Moreover, X-ray microscopes that exploit the computer-tomog- raphy (CT) method can image the three-dimensional (3D) structure of diverse samples [12–16]. However, these methods require large and complex synchrotron light sources. Furthermore, 3D reconstructions using these systems require the acquisition of many images with the sample rotated and/or tilted between each image, and acquiring these numerous images can lead to sample damage. Moreover, to acquire an image at each viewing angle, the beam focus and the sample position must be adjusted between each image, which leads to long data-acquisition times. Recently, we have developed a soft-X-ray microscope called the scanning-electron generation X-ray microscope (SGXM) [17–19]. This microscope consists of a simple system for detecting the soft X-rays generated by the interaction between a scanning electron beam of a scanning-electron microscope (SEM) and a target material layer on thin film. Therefore, X-ray imaging of various samples is relatively simple with SGXM. Furthermore, the spatial resolution of the SGXM incorporated into a field-emission SEM is 11 nm [19] and its theoretical resolution is less than 5 nm [18]. In this paper, we present a 3D reconstruction system based on the SGXM technology for studying atmospheric biological samples. The X-ray detection system consists of a linear X-ray photodiode (PD) array; hence, multiple sample tilt images are acquired simultaneously by PDs during a single electron-beam (EB) scan. Moreover, we have developed a highly accurate 3D reconstruction method that uses a simulated-annealing (SA) algorithm. Currently available methods for 3D volume reconstruction include the filtered-back projection method, the algebraic reconstruction technique and the simultaneous iterative recon- struction technique [20,21]. However, these methods require numerous projections. Some two-dimensional (2D) CT methods have been developed that use the SA algorithm and enable highly accurate reconstructions from a reduced number of projections [22–24]. Furthermore, we have developed a 3D reconstruction method that uses an SA algorithm and have applied it to imaging proteins with a transmission electron microscope [25]. We, thus, propose herein a 3D reconstruction method that uses an SA algorithm combined with images captured by a linear array of X-ray PDs. The atmospheric sample holder, the linear-array X- ray PD system and the SA 3D reconstruction algorithm constitute a new technology that is expected to contribute in a broad range of scientific fields for analysing the 3D structure of various atmospheric samples. Results A schematic of the atmospheric 3D-SGXM system is shown in Figure 1. The unstained sample is positioned under a brass PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e21516