Fig. 1

Schematic describing confocal imaging and processing technique. a Physical positioning of femur during imaging. b Subset of confocal image sections taken of the dome of a medial femoral condyle, looking from the apex downward in the z-direction (image inverted for clarity). c Region of interest (rectangle) selected from the 3D reconstruction of the condyle surface. d The method used to differentiate space occupied by cells and extracellular matrix. Areas of cellularity were labeled with “1” and non-cell areas were labeled with “0”. Black bars represent the vertical distance between areas of cellularity within the most superficial and deepest confocal images. Vertical distances were averaged across the width of the section to estimate the thickness of the superficial and upper intermediate zone. e Schematic demonstrating the rectangular prisms created iteratively and used to approximate the volume of the superficial and upper intermediate zone