Observation of Atmospheric Samples by 3D-SGXM Figure 5. Evolution in 3D reconstruction and projection of XPD-4 at annealing cycles. (A) Evolution of 3D structure as a function of the number of annealing cycles specified above each 3D image. Initially, the 3D volume is blank. Starting from the blank volume, the 3D volume rapidly emerges in just 10 cycles. Beyond 10 cycles, the 3D volume is gradually optimized and the fine structure appears. (B) Evolutions of the projection image corresponding to XPD-4 angle (4.4u) after the specified number of annealing cycles. (C) Error and temperature as a function of annealing cycles. The temperature remains constant at 10 for the first 30 cycles. After 30 cycles, the temperature decreases exponentially. However, within 10 cycles, the error is rapidly reduced. After 10 cycles, the error decreases gradually. doi:10.1371/journal.pone.0021516.g005 Devices, Inc., Japan). The sputtering conditions were 1.1 Pa Ar, 150 mA and 30 s sputtering time. The distance between the sputter target and the Si3N4 film was 50 mm. The deposited CuZn layer was approximately 60 nm thick, as determined by the sputter conditions. Sample preparation and an atmospheric sample holder The biological sample of Rhodobacter capsulatus, obtained from the Shimatec Co. (Japan, Osaka), is a purple, rod-shaped, nonsulfur photosynthetic bacterium with flagella [26,27]. One millilitre of bacterium solution was centrifuged at 6200 rpm for 1 min in a Capsulefuge PMC-060 (Tomy, Inc., Japan), and the supernatant solution was replaced by a 1% (w/v) trehalose solution of 1 ml (Hayashibara, Inc., Japan). To prepare the SEM sample, 2 ml of the bacterium solution was dropped onto a CuZn-coated Si3N4 film. After 1 min, the solution on the film was removed by filter paper and the film was dried in atmosphere at room temperature (23uC) for 5 min. The sample thus attached to the Si3N4 film was secured in the atmospheric sample holder (Figure 1). The new sample holder maintains the sample at atmospheric pressure in the sample space between a CuZn-coated 50-nm Si3N4 film and a 100-nm Si3N4 film (Figure 1A). The sample space between the two Si3N4 films is sealed by two sample-holding pieces and two O-rings, and the holding pieces are coupled by screws. The biological sample in the atmospheric sample holder is then mounted onto the sample stage above the linear PD array (Figure 1B and D). Scanning electron microscopy and X-ray imaging system The stage containing the sample was transferred to the chamber of a thermionic emission SEM (JSM-6390, JEOL, Japan). X-ray images were captured by an original SE detector and the linear X- ray PD array under high-vacuum conditions with the following parameters: magnification = 4000–30,0006, image size = 12806 960 pixels, observation time = 160 s, working distance = 7 mm, EB accelerating voltages = 5 kV and EB aperture = 42–45. For the bacterium sample imaged at 4000 and 30,0006 magnifications (Figure 2), the X-ray images from the SE detector were filtered by a 2D Gaussian filter (GF) (size = 969 pixels, s = 1) by using Matlab R2007b (Math Works Inc., USA). The image contrast level was normalized to black (low intensity) and white (high intensity). A linear X-ray PD array model AXUV-16EL (IRD Inc., USA) was fixed under the sample and consisted of seven PD elements out of the possible 16 elements (Figure 1). The XPD output was recorded using a data recorder (EZ7510, NF Co., Japan) after amplification by PD preamplifiers (gain = 1006 MA/V) and the main amplifiers (gain 1006), which consist of AD8562 (Analog devices Inc., USA) integrated circuits. The X-ray and EB scan signals were recorded in a 16-bit data recorder with 20-kHz sampling rate. After the experiments, the data files were transferred to a personal computer (Intel Core2 Duo E6850, 3.0 GHz, Windows XP), and the X-ray images were generated from the XPD signals combined with the EB scan signal, with the calculation performed by Matlab R2007b. PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e21516