Second harmonic generation imaging microscopy

University Of Connecticut Sch Of Med/Dnt, Farmington CT

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Abstract

Type I collagen is the most abundant extra-cellular matrix protein being the primary structural component of bone, skin tendon and to a lesser extent blood vessels. Although its biochemical and ultrastructural properties are well understood, there are still gaps in our knowledge the precludes genotype/phenotype predictions in diseases affecting its primary structure or its gene regulation. This proposal will evaluate a new method for visualizing extra-cellular matrix Type I collagen fibers in cell culture or in intact tissues in real time utilizing second harmonic generation (SHG) imaging microscopy. This is a new and novel imaging technique that can be used to probe endogenous structural proteins with high contrast without the need for exogenous labels. Furthermore, this method provides intrinsic 3-dimensionality and is ideal for intact tissues since samples (up to 1 mm) can be imaged in situ. This method provides resolution on the order of 500 nm and is ideal for probing higher-level organization of collagen fibrils and fibers. SHG imaging will allow the direct visualization of some of the physical properties of the collagen triple helices. Studying the organization of collagen fibrillar structure by SHG imaging will in fact complement existing ultrastructural (X-ray diffraction and electron microscopy) and non-imaging (NMR, CD) methods. The value of this approach will be assessed in two disease of type I collagen that are a consequence of either under accumulation (osteogenesis imperfecta, OI) or excessive accumulation (tight skin, Tsk) of type I fibers in bone or skin respectively. In both cases, the relationship between the underlying genetic abnormality and the severity of the disease is not understood and a unique insight into this relationship may come from this method. We will first establish quantitative measurements of the collagen fibers in the ECM of normal murine tissues by SHG where we will utilize both tissue culture cells, and as well as tissues and decalcified bones from wild type mice. This methodology may ultimately become a powerful clinical tool both in terms of predictive value of musculo-skeletal diseases as well as in the assessment of the efficacy of drug and gene therapies.