TIME-LAPSE CONFOCAL REFLECTION (TLR) IMAGING OF EXTRACELLULAR MATRIX (ECM) ASSEMBLY IN VITRO
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A.O. Brightman1, B.P. Rajwa2, J.E. Sturgis2, M.E. McCallister1, J.P. Robinson2,
1Dept. of Biomedical Engineering,
Time-Lapse Reflection Imaging of Matrix Assembly - Collagen, type I, or pepsin-solubilized intestinal submucosal, PSIS, was polymerized in 8-well chambered coverglass (Lab-Tek, Nalge Nunc Int.). The unstained 3-dimensional matrices were viewed with an inverted confocal laser-scanning microscope (BioRad MRC1024) using a 60X, oil immersion lens. Laser light of 488 nm illuminated the hydrated samples and the reflected light was detected with a photomultiplier tube using a blue reflection filter. Samples were maintained at 30-37°C on a heated stage. Time-lapse images of fibrillogenesis were recorded at 10-15 second intervals. For standardizing the scanning depth in the matrix, the Z position was kept in the range of 20 to 50 µm from the coverglass.
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