e. around 1 Å for hard X-rays. Ptychographic CDI is an emerging iterative phase retrieval method with no fundamental limitation in sample size, which provides the complex sample transmission function. Ptychographic CDI has recently been combined with a CT setup for ptychographic (X-ray) CT, where the LCN of the

femoral mid-diaphysis in the mouse has been retrieved at an isotropic voxel size of 65 nm [26], offering a continuous representation of individual canaliculi. In addition to the reconstructed LCN morphology, the local mineral density was simultaneously reconstructed in the same experiment by ptychographic CT in terms of (absolute) electron density with fluctuations of less than 0.2% corresponding to less than 5 mg/cm3 in mass density. A key problem, which is common to all the CT-based techniques described above, CX-5461 mouse is their limited field of view (FOV) which adversely affects the assessment of larger tissue volumes, containing for example a representative segment of the osteocyte network and/or the LCN. One concept to overcome this limitation in 3D

at a sufficiently high resolution is the strategy to go back to the elementary direct imaging method of consecutive physical probe sectioning and imaging, similar to conventional histology based on light microscopy. Selleckchem PD0325901 However, the imaging approach must have improved spatial resolution compared to light microscopy and it must be automated in order to resolve the intracortical and intratrabecular PIK-5 bone microstructure in a relevant volume. One implementation of this concept is serial focused ion beam/SEM (FIB/SEM). In serial FIB/SEM, several thin sections in the 10 nm range are milled away from the sample’s block face using a focused ion beam, which replaces the diamond knife for mechanical cutting in traditional histology. These sections are then scanned

by SEM. When applied in a serial and automated fashion, a 3D reconstruction of the specimen can be generated at EM resolution. FIB systems have been mostly used in materials science and in the semiconductor industry since the early 1980s, and the application of serial FIB/SEM has broadened with the automation of the dual beam FIB/SEM imaging process in the mid-2000s, including research fields in the life sciences, especially in the neurosciences [27]. Regarding hard tissue characterization, serial FIB/SEM has been broadly employed to study dental/implant interfaces and bone/implant interfaces. Moreover, Earl et al. lately examined the intradental tubule network, including major, fine, and microbranches from the micrometer range down to several hundred nanometers in diameter [28], similar to the dimensions of the canaliculi in bone. The first attempt to image the LCN in bone goes back to Stokes et al. [29], where the representation of the canaliculi (species not specified) was fragmentary only. More recently, Schneider et al.