(CuZn)-coated Si3N4 film and sealed by a 100-nm Si3N4 film (Figure 1A), and the ensemble is attached to an atmospheric sample holder (Figure 1B) in which the sample space is maintained at atmospheric pressure. The sample holder is then mounted on the stage of the linear X-ray PD array system combined with a photoelectric conversion film (Figure 1B–D). The CuZn-coated Si3N4 film is scanned by an EB accelerated at a voltage of 5 kV; hence, the impinging electrons are almost absorbed on the CuZn-coated Si3N4 film (Figure S1). Conse- quently, electron damage to the sample is strongly suppressed. In addition, the interaction of the scanning EB with the CuZn thin layer emits approximately 1-keV X-rays (Figure S1) that irradiate the sample. The X-rays transmitted through the sample are detected by the linear PD array and photoelectron conversion systems (Figure 1B). To form X-ray images, photoelectrons from the Au-coated film are detected by a secondary-electron (SE) detector in the SEM, which is typically supplied with conventional SEMs. This process constitutes the photoelectron conversion system [19]. As a result, X-ray images can easily be observed because the same SEM position setting and focus can be used. To form X-ray images from the linear PD array, the X-ray signals are read from the seven PDs that are used from among the 16 elements at one PD interval (Figure S2). The seven PD outputs are amplified by preamplifiers and main amplifiers and then recorded with a data recorder (Figure 1B). Because of their linear arrangement, each PD views the sample from a different angle and Observation of Atmospheric Samples by 3D-SGXM hence collects a different tilt image. The angles between the EB- irradiated spot in the Si3N4 film and the PD elements range from 240.2u to 45.0u (Figure S2). Finally, the X-ray images are reconstructed from the X-ray signals combined with the EB scan signal and the PD angle corrections. With the 3D-SGXM system, we first observed unstained atmospheric bacteria of Rhodobacter capsulatus [26,27]. Figure 2A shows an X-ray image of the bacteria positioned under the CuZn- coated Si3N4 film from the SE detector, acquired at 40006 magnification at a 5-kV EB. The unstained bacteria create a black contrast and have an elongated shape, which is 2 to 5 mm long and approximately 1 mm wide. The bacterium indicated by the white arrow in Figure 2A was scanned at 30,0006 magnification and is shown in the top-left panel of Figure 2B. The inner structure of this bacterium is clearly visible at this magnification. Figure 2B shows the simultaneously acquired tilt images of the same bacterium obtained from the linear PD elements and corrected for angle and X-ray absorbance. In these images, X-ray PD-1 (XPD-1) views the sample at h1 = 240.2u; XPD-4 is placed approximately under the sample (h4 = 4.4u) and XPD-7 views the sample at h7 = 45u (Figure S2). To correct the tilt angle, the horizontal axes of the detected images are reduced by a factor cos(hx). Furthermore, in the 3D model, X-ray images are converted to X-ray absorbance after angular correction (see Materials and Methods). To construct a high-accuracy 3D model from seven X-ray images, we developed a new 3D reconstruction method that uses Figure 1. Experimental setup and new 3D-SGXM system. (A) Schematic of the atmospheric sample holder. Bacteria samples are positioned under the CuZn-coated Si3N4 film; these samples are maintained at atmospheric pressure. A scanning 5-kV EB irradiates the upper side of the CuZn- coated Si3N4 film. The linear X-ray PD array under the sample detects the X-rays transmitted through the sample. (B) Schematic of X-ray-detection apparatus. The X-ray images are acquired by both the SE detector (which detects the photoelectric conversion electrons) and the linear X-ray PD array. The X-ray image acquired from the SE detector was obtained from the photoelectrons generated at the Au-coated film, which is positioned at the side of the PD stage. A linear X-ray PD array consisting of seven PDs was fixed under the sample, and its output was recorded using a data recorder (EZ7510, NF Co., Japan) after amplification by PD preamplifiers (gain 1006 MA/V) and main amplifiers (gain 1006). (C) Photograph of the linear X-ray PD array system. The linear X-ray PD array is visible in the upper centre on a circuit board. In this system, the X-ray signals are obtained by seven X-ray PDs (XPD 1–7) from among the 16 elements, and the output from each X-ray PD is amplified by a preamplifier, which is located under the circuit board. (D) Photograph of atmospheric sample holder set upon the X-ray detection system. The linear X-ray PD array system is covered by an Al box, but its position is marked by the hole that is visible beneath the sample holder. An atmospheric sample holder was mounted on the Al stage. The Au-coated photoelectron conversion film is fixed in the gap between the sampler holder and the X-ray detection box. doi:10.1371/journal.pone.0021516.g001 PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e21516