Observation of Atmospheric Samples by 3D-SGXM Figure 3. Outline of new 3D reconstruction method that uses SA algorithm. First, a 3D mask is calculated from the X-ray images of the sample. Next, a position in the 3D mask is selected at random and its voxel values within the kernel-boundary area are altered at random. The thus- modified 3D volume is reprojected onto the seven angular directions to the X-ray detectors. To evaluate the modified values, the residual sum of the squares between all projections and its corresponding X-ray images is calculated and used by the SA algorithm to determine whether to accept the new 3D volume. This procedure is iterated 100,000 times, and when finished, the temperature, the kernel size and its value-shifting SD decrease exponentially. These processes are iterated for a prefixed cycles. doi:10.1371/journal.pone.0021516.g003 already approximates the X-ray images. After 200 cycles, the projection is very similar to the corresponding X-ray image. Figure 5C shows the annealing temperature and the error between the projections and the X-ray images as functions of the cycle number. Between 0 and 10 cycles, the error decreases drastically and then it decreases gradually with the cycle number. Figure 6 shows the projections and the 3D volume once annealing is complete. Each model projection shown in Figure 6A is very similar to the X-ray images for the corresponding tilt angle (Figure 2B). Therefore, the calculated 3D volume is likely to be highly reliable. The 3D volume viewed from the side and the top and the corresponding cross sections are shown in Figure 6B–E; Figure 6B and C show that the bacterium has the form of a slightly curved cylinder. A higher-density area in the bacterium, which is probably the nucleoid, is located at the bottom right, and the point with slightly above-average density is detected at the top left in the bacterium (Figure 6D and E). The bacterium centre exhibits a complex grain structure with relatively low density. To clearly identify the high-density region, we captured images of three bacteria of similar length and have displayed their inner structures in Figure 7. Figure 7A shows the same bacterium that is displayed in Figure 6 and reveals that the red high-density regions are clearly located in the longitudinal extremities of the bacterium. Furthermore, the complex inner structure of this bacterium is clearly observed. These features of the high-density regions shown in Figure 7A are similar to those observed in the other two bacteria shown in Figure 7B and C. Discussion The results presented above demonstrate that the proposed system (Figures 1–4) can obtain a high-resolution 3D structure of the specimens in an atmospheric unstained sample (Figures 5–7). These images clearly reveal the complex structure of the high- density regions of the bacteria. The enabling technology is the linear array of X-ray PD elements that simultaneously capture a sequence of tilt X-ray images without requiring sample rotation or tilt (Figures 1 and 2). Therefore, the proposed system can calculate the 3D volume at the point of sample fixing, which enables high- speed 3D detection. In addition, this aspect of the technology further contributes to reducing sample damage. The spatial resolution of the proposed system can be estimated from the geometry of the X-ray source diameter, the PD size and the separation between the two [18]. X-ray detector XPD-4 is positioned directly under the sample at a distance of 13 mm and presents a rectangular cross section of 2 mm65 mm (in the x and y directions, respectively, Figure S4A). For a 5-kV EB acceleration voltage, a Monte Carlo simulation using CASINO version 2.42 [28] (Figure S4B and C) indicates an X-ray source diameter of approximately 25 nm. The area of the sample covered by the X- ray beam expands in proportion to the depth from the X-ray emission point in the CuZn-coated film (Figure S4A). The spatial resolution of the image captured by XPD-4 is 78 nm6160 nm for a bacterium centred (z = 300 nm depth) (Figure S4D). At the right side of the PD array, XPD-7 views the sample at 45u with respect to EB (Figure S4A) and is 18.4 mm from the sample. Its resolution is 52 nm692 nm at a depth of 300 nm, which is superior to the resolution of XPD-4. However, the X-ray image generated by XPD-7 is noisy because it is farther from the sample than XPD-4. Accordingly, more sensitive X-ray detectors with smaller cross sections are required to improve the 3D resolution beyond what is reported here. Furthermore, because it has many X-ray detectors, the 2D PD array is a suitable geometry for generating the high- resolution 3D model. PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e21516