Fig. 8

Necrotic cartilage canals associated with necrotic chondrocytes. Cyan: Two-photon-excited fluorescence (TPEF), Glowing red: Second harmonic generation (SHG). a An area with necrotic cells (indicated by dashed lines) is associated with a necrotic cartilage canal that enters the epiphyseal growth cartilage from the secondary ossification centre. The canal is surrounded by foci of intense SHG signal, associated with both necrotic and viable chondrocytes. Near the ossification front, the canal is narrowed. b The magnified TPEF-image shows the difference in cell size between the necrotic and viable chondrocytes. c A corresponding H&E-stained sample of an adjacent section verifies that the cell morphology can be visualized using TPEF. Some lacunae appear empty due to cell loss during sectioning. d A necrotic and dilated cartilage canal (asterisk) is associated with an area of necrotic chondrocytes (indicated by dashed lines). The canal originates from the caudal cruciate ligament (CCL) attachment site. The collagen fibres of the ligament give intense SHG signal. Accumulation of erythrocytes is observed within the necrotic canal. 10 x-focusing objective was used in a, while a 25×–objective was used in b. The bars are equal to 500 μm in a and d and 50 μm in b and c