Determination of Interstitial Collagen Deposition and Related Topography Using the Second Harmonic Generation-Based...

Interstitial fibrosis is characterized by the increased deposition of extracellular matrix (ECM) components within the interstitial space of various organs, such as the kidneys, heart, lungs, liver, and skin. The primary component of interstitial fibrosis-related scarring is interstitial collagen. Therefore, the therapeutic application of anti-fibrotic medication hinges on the accurate measurement of interstitial collagen levels within tissue samples. Current histological measurement techniques for interstitial collagen are generally semi-quantitative in nature and only provide a ratio of collagen levels within tissues. However, the Genesis™ 200 imaging system and supplemental image analysis software, FibroIndex™, from HistoIndex™, is a novel, automated platform for imaging and characterizing interstitial collagen deposition and related topographical properties of the collagen structures within an organ, in the absence of any staining. This is achieved by using a property of light known as second harmonic generation (SHG). Using a rigorous optimization protocol, collagen structures in tissue sections can be imaged with a high degree of reproducibility and ensures homogeneity across all samples while minimizing the introduction of any imaging artefacts or photobleaching (decreased tissue fluorescence due to prolonged exposure to the laser). This chapter outlines the protocol that should be undertaken to optimize HistoIndex scanning of tissue sections, and the outputs that can be measured and analyzed using the FibroIndex™ software.