Observation of Atmospheric Samples by 3D-SGXM Figure 4. Flowchart of newly developed 3D reconstruction method employing SA algorithm. First, the 3D mask of the sample is calculated from the tilt X-ray images acquired by the linear X-ray PD array. Next, we select a random position in the 3D mask, change its voxel values in a random shift, calculate the new error from the new 3D volume and feed this new error to the SA algorithm to determine whether to accept the modification. The error value used to determine whether to accept the modification is the residual sum of the squares of the projections from the new 3D volume and its corresponding X-ray images. This process is iterated for a predetermined number of times (100,000 times for the work reported herein). The temperature and kernel of changing voxels are thereby gradually reduced. These steps are iterated for a predetermined number of cycles (200 cycles for the work reported herein). doi:10.1371/journal.pone.0021516.g004 In the future, the system should be able to produce real-time 3D models using highly sensitive 2D X-ray detectors. Furthermore, work is in progress to develop an easy-to-use water-sample holder. A combination of these systems is expected to enable high- resolution observation of real-time movements of biological samples in water. In conclusion, we have developed an X-ray CT system called 3D-SGXM that enables the observation of the 3D structure of atmospheric unstained specimens. The specimens are placed under a CuZn-coated Si3N4 film and the ensemble is secured in an atmospheric sample holder, so that the samples are in an atmospheric environment. Tilt X-ray images of the sample are detected by a linear X-ray PD array, and the sample’s 3D structure is generated by a newly developed 3D reconstruction method that uses an SA algorithm. The 3D reconstructions presented herein clearly reveal the inner structure of the bacterium samples. This method can be easily used for diverse samples in a broad range of scientific fields. Materials and Methods CuZn coating on Si3N4 film A 50-nm-thick Si3N4 film supported by a window (0.5 mm60.5 mm) in a Si frame (5 mm65 mm and 0.2 mm thick; Silson Ltd., UK) was coated with brass (70% Cu, 30% Zn) using a magnetron sputter machine model MSP-30T (Vacuum PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e21516