Observation of Atmospheric Samples by 3D-SGXM Figure 2. X-ray image of atmospheric unstained bacteria by SGXM system. (A) X-ray image of the atmospheric Rhodobacter capsulatus acquired by the photoconversion system using an original SE detector. The image was acquired at 40006 magnification at a 5-kV EB acceleration voltage. The image was filtered by a 2D Gaussian filter (size 15615 pixels; s = 1). (B) High-resolution X-ray images of the bacterium indicated by white arrow in panel (A). The top left image shows an X-ray image of a bacterium acquired with the SE detector and taken at 30,0006magnification. The X- ray images from XPD 1–7 were acquired by the linear PD array. Each X-ray image was corrected for the view angle and X-ray absorbance. In these images, XPD-1 is the tilt image at 240.2u, XPD-4 is placed approximately under the sample (4.4u) and XPD-7 is at 45u. doi:10.1371/journal.pone.0021516.g002 the SA algorithm (Figures 3 and 4). This algorithm enables 3D reconstruction from a small number of projections, even if its projections are restricted tilt angles. A schematic of the proposed 3D reconstruction method is shown in Figure 3 and its flow diagram is given in Figure 4. Initially, a 3D mask is calculated by back projecting the 2D masks obtained from the X-ray images (Figure S3). The 3D voxel values and the seven projections are initially set to 0. Next, a position in the 3D mask is randomly selected and its voxel values within the kernel-boundary area are randomly shifted. This new 3D volume is reprojected onto the seven angles of the corresponding PDs. To evaluate the modified voxel values, the residual sum of the squares between all reprojections and its corresponding X-ray images is calculated and used by the SA algorithm to determine whether to accept the new 3D volume (Figure 4). This procedure is iterated 100,000 times. When finished, the temperature, the kernel size and is the standard deviation (SD) decrease exponentially. The kernel area begins with an SD of 15s voxels, which is finally reduced to 2s voxels. The entire cycle is repeated 200 times. We applied our 3D reconstruction method to the tilt X-ray images of a bacterium sample (Figure 2B). The modifications in the 3D volume and the XPD-4 model projections between various annealing cycles are shown in Figure 5A and B, respectively. Starting from the blank volume and its projected image (left-most panels in Figure 5A and B), the 3D volume and the projection rapidly emerge after just 10 cycles, at which point the projection PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e21516