Multiphoton and SHG signals originating from tendon histological preparations. MPEF images show general tissue morphology of tendon and associated structures, while spectrally clean SHG images specifically reveal collagen present in the same region. Representative SHG (A,D), MPEF (B,E) or combined images (C,F,I) obtained from standard histological thin tissue section are shown. In normal healthy tendon (A-C), SHG demonstrates tightly bundled, longitudinally oriented collagen fibres. Tenocytes (B, C, asterisk) are inconspicuous due to their sparse cytoplasm. In early tendinosis tendon, collagen surrounding abnormal tenocytes (D-F) demonstrates a disturbed organization, and the average SHG signal is greatly reduced (c.f. panel A). The tenocytes in tendinosis tendon demonstrate much more prominent cytoplasm and a rounded, as opposed to spindle-shaped, morphology (E). Picrosirius red-stained tendon from tendinosis tendon (G, brightfield; H, polarized light) is shown as a comparison. Tendinosis regions demonstrate a loss of birefringence (H). In comparison, SHG signal reveals the presence of abnormal pericellular fibrillar collagen structures not visualized with polarized light. Scale bars represent 25 μm for A-H, and 10 μm for I.