Fig. 3

Cartilage canal with a boundary of collagen fibres. Cyan: Two-photon-excited fluorescence (TPEF), Glowing red: Second harmonic generation (SHG). a Cross-section of a cartilage canal surrounded by foci of intense SHG. b Hematoxylin and eosin-stained sample showing the same canal as in (a) but from an adjacent section. The canal has a margin that stains intensely eosinophilic (arrow). c A part of the canal (region indicated with the dashed rectangle in (a)) at higher magnification. Strands of high SHG intensity, indicating collagen fibres, appear to originate in the interior of the canal and align at the canal margin. d The TPEF signal gives the morphology of the various cell types at the both sides of the boundary between the cartilage canal and the adjacent cartilage. The interior of the canal contains cells with long processes (asterisk). At the boundary, the cell shape becomes flat, and in the adjacent cartilage, the cells are gradually more spherical. 10×–focusing objective was used in a and b, and 63× was used in c and d. All bars equal 100 μm