A novel workflow for multi-modal imaging of musculoskeletal tissues.

According to the World Health Organization (WHO) musculoskeletal conditions are a leading contributor to disability worldwide. This fact is often somewhat overlooked, since musculoskeletal conditions are less likely to be associated with mortality. Nonetheless, treatments, therapies and management of these conditions are extremely costly to national healthcare systems. As with all systemic conditions, biomedical imaging of relevant tissues plays a major role in understanding the fundamental biological processes involved in musculoskeletal health. However, the skeletal system with its relatively large proportion of dense, opaque (often mineralised) tissues can often be more challenging to image, and recently important advances have been made in imaging these complex musculoskeletal tissues. Thus, we here describe a novel workflow in which recent advanced imaging techniques have been modified and optimised for use in musculoskeletal tissues (specifically bone and cartilage). This will allow for investigations, of different phases of these tissues, at new and higher resolutions. Furthermore, the process has been designed to fit with the existing and standard processes which are typically used with these samples (i.e. μCT imaging and standard histology). The additional modalities which have been included here are second harmonic generation (SHG) imaging, tissue clearing, specifically the Passive Clear Lipid-exchanged Acrylamide-hybridised Rigid Imaging Tissue hYdrogel (CLARITY) method known as PACT, and then imaging of these tissues with confocal, multiphoton and light-sheet microscopy. This paper serves to introduce a combination of existing new methods and improvements in imaging of musculoskeletal tissues.