Multiplexed Imaging of Osteocytes in Bone

Osteocytes are embedded in bone and account for 90% of cells within mature bone. Although previously viewed as quiescent cells, recent research has revealed the importance of osteocytes in regulation of bone remodeling, both as a mechanosensor and a source of signaling molecules for regulating osteoclasts and osteoblasts. Many osteocyte imaging techniques rely on imaging the lacunocanalicular space around the cell rather than directly imaging osteocytes themselves. To better characterize differences in osteocyte morphology and structure in aging and disease, we have developed a multiplexed imaging technique with the goal of examining osteocytes in 3D within their bone environment. 50-100 micron-thick decalcified bone sections were prepared and a variety of imaging dyes were used in combination with confocal microscopy to image bone matrix, lacunocanalicular space, osteocyte cell membrane, nuclei, and cytoskeleton in various combinations. We have simultaneously imaged collagen matrix (using a transgenic mouse line expressing GFP-tagged-collagen), lacunocanalicular space (using a fixable Texas Red-labeled dextran), osteocyte cytoskeleton (using alexa-647-phalloidin) and nuclei (using DAPI). We have also employed DiO labeling of the cell membrane in addition to dextran labeling of the lacunocanalicular system and DAPI imaging of cell nuclei. DiI labeling of the membrane in combination phalloidin labeling of the cytoskeleton was problematic in bone slices with either incomplete penetration of DiI in the sample or loss of phalloidin specificity. Mounting of bone slices in 2-2'-thiodiethanol increased imaging depth and resolution but resulted in loss of GFP and phalloidin signal over time, therefore requiring imaging immediately after mounting. From multiplexed confocal image stacks it is possible to simultaneously study several aspects of osteocyte structure in demineralized bone matrix with sufficient resolution to render detailed 3-D reconstructions of imaged volumes. These methods can be applied to studying osteocyte structure/morphology in normal and diseased bone tissues.