Minimally invasive longitudinal intravital imaging of cellular dynamics in intact long bone.

Intravital two-photon microscopy enables deep-tissue imaging at high temporospatial resolution in live animals. However, the endosteal bone compartment and underlying bone marrow pose unique challenges to optical imaging as light is absorbed, scattered and dispersed by thick mineralized bone matrix and the adipose-rich bone marrow. Early bone intravital imaging methods exploited gaps in the cranial sutures to bypass the need to penetrate through cortical bone. More recently, investigators have developed invasive methods to thin the cortical bone or implant imaging windows to image cellular dynamics in weight-bearing long bones. Here, we provide a step-by-step procedure for the preparation of animals for minimally invasive, nondestructive, longitudinal intravital imaging of the murine tibia. This method involves the use of mixed bone marrow radiation chimeras to unambiguously double-label osteoclasts and osteomorphs. The tibia is exposed by a simple skin incision and an imaging chamber constructed using thermoconductive T-putty. Imaging sessions up to 12 h long can be repeated over multiple timepoints to provide a longitudinal time window into the endosteal and marrow niches. The approach can be used to investigate cellular dynamics in bone remodeling, cancer cell life cycle and hematopoiesis, as well as long-lived humoral and cellular immunity. The procedure requires an hour to complete and is suitable for users with minimal prior expertise in small animal surgery.

Apoptotic cell fragments locally activate tingible body macrophages in the germinal center.

Germinal centers (GCs) that form within lymphoid follicles during antibody responses are sites of massive cell death. Tingible body macrophages (TBMs) are tasked with apoptotic cell clearance to prevent secondary necrosis and autoimmune activation by intracellular self antigens. We show by multiple redundant and complementary methods that TBMs derive from a lymph node-resident, CD169-lineage, CSF1R-blockade-resistant precursor that is prepositioned in the follicle. Non-migratory TBMs use cytoplasmic processes to chase and capture migrating dead cell fragments using a "lazy" search strategy. Follicular macrophages activated by the presence of nearby apoptotic cells can mature into TBMs in the absence of GCs. Single-cell transcriptomics identified a TBM cell cluster in immunized lymph nodes which upregulated genes involved in apoptotic cell clearance. Thus, apoptotic B cells in early GCs trigger activation and maturation of follicular macrophages into classical TBMs to clear apoptotic debris and prevent antibody-mediated autoimmune diseases.