Nonlinear optical microscopy: use of second harmonic generation and two-photon microscopy for automated quantitative liver fibrosis studies

Liver fibrosis is associated with an abnormal in- crease in an extracellular matrix in chronic liver diseases. Quantitative characterization of fibrillar collagen in intact tissue is essential for both fibrosis studies and clinical ap- plications. Commonly used methods, histological staining followed by either semiquantitative or computerized im- age analysis, have limited sensitivity, accuracy, and operator-dependent variations. The fibrillar collagen in si- nusoids of normal livers could be observed through second-harmonic generation SHG microscopy. The two- photon excited fluorescence TPEF images, recorded si- multaneously with SHG, clearly revealed the hepatocyte morphology. We have systematically optimized the pa- rameters for the quantitative SHG/TPEF imaging of liver tissue and developed fully automated image analysis algo- rithms to extract the information of collagen changes and cell necrosis. Subtle changes in the distribution and amount of collagen and cell morphology are quantita- tively characterized in SHG/TPEF images. By comparing to traditional staining, such as Masson's trichrome and Sirius red, SHG/TPEF is a sensitive quantitative tool for automated collagen characterization in liver tissue. Our system allows for enhanced detection and quantification of sinusoidal collagen fibers in fibrosis research and clini- cal diagnostics. © 2008 Society of Photo-Optical Instrumentation Engi-