Measuring collagen fibril diameter with differential interference contrast microscopy

Collagen fibrils, linear arrangements of collagen monomers, 20-500 nm in diameter, comprising hundreds of molecules in their cross-section, are the fundamental structural unit in a variety of load-bearing tissues such as tendons, ligaments, skin, cornea, and bone. These fibrils often assemble into more complex structures, providing mechanical stability, strength, or toughness to the host tissue. Unfortunately, there is little information available on individual fibril dynamics, mechanics, growth, aggregation and remodeling because they are difficult to image using visible light as a probe. The principle quantity of interest is the fibril diameter, which is difficult to extract accurately, dynamically, in situ and non-destructively. An optical method, differential interference contrast (DIC) microscopy has been used to visualize dynamic structures that are as small as microtubules (25 nm diameter) and has been shown to be sensitive to the size of objects smaller than the wavelength of light. In this investigation, we take advantage of DIC microscopy's ability to report dimensions of nanometer scale objects to generate a curve that relates collagen diameter to DIC edge intensity shift (DIC-EIS). We further calibrate the curve using electron microscopy and demonstrate a linear correlation between fibril diameter and the DIC-EIS. Using a non-oil immersion, 40x objective (NA 0.6), collagen fibril diameters between ~100 nm to ~ 300 nm could be obtained with ±11 and ±4 nm accuracy for dehydrated and hydrated fibrils, respectively. This simple, nondestructive, label free method should advance our ability to directly examine fibril dynamics under experimental conditions that are physiologically relevant.