Automatic image acquisition, calibration and montage assembly for biological X‐ray microscopy

We describe a system for the automatic acquisition and processing of digital images in a high‐resolution X‐ray microscope, including the formation of large‐field high‐resolution image montages. A computer‐controlled sample positioning stage provides approximate coordinates for each high‐resolution subimage. Individual subimages are corrected to compensate for time‐varying, non‐uniform illumination and CCD‐related artefacts. They are then automatically assembled into a montage. The montage assembly algorithm is designed to use the overlap between each subimage and multiple neighbours to improve the performance of the registration step and the fidelity of the result. This is accomplished by explicit use of recorded stage positions, optimized ordering of subimage insertion, and registration of subimages to the developing montage. Using this procedure registration errors are below the resolution limit of the microscope (43 nm). The image produced is a seamless, large‐field montage at full resolution, assembled automatically without human intervention. Beyond this, it is also an accurate X‐ray transmission map that allows the quantitative measurement of anatomical and chemical features of the sample. Applying these tools to a biological problem, we have conducted the largest X‐ray microscopical study to date.