Polarized light microscopy, variability in spider silk diameters, and the mechanical characterization of spider silk

Spider silks possess a remarkable combination of high tensile strength and extensibility that makes them among the toughest materials known. Despite the potential exploitation of these properties in biotechnology, very few silks have ever been characterized mechanically. This is due in part to the difficulty of measuring the thin diameters of silk fibers. The largest silk fibers are only 5–10 mm in diameter and some can be as fine as 50 nm in diameter. Such narrow diameters, coupled with the refraction of light due to the anisotropic nature of crystalline regions within silk fibers, make it difficult to determine the size of silk fibers. Here, we report upon a technique that uses polarized light microscopy (PLM) to accurately and precisely characterize the diameters of spider silk fibers. We found that polarized light microscopy is as precise as scanning electron microscopy (SEM) across repeated measurements of individual samples of silk and resulted in mean diameters that were B0.10 mm larger than those from SEM. Furthermore, we demonstrate that thread diameters within webs of individual spiders can vary by as much as 600%. Therefore, the ability of PLM to non-invasively characterize the diameters of each individual silk fiber used in mechanical tests can provide a crucial control for natural variation in silk diameters, both within webs and among spiders. Additional key words: major ampullate, flagelliform, fibers, tensile test, orb web Spider silks are among the strongest and toughest fibers known to science. Using a diverse array of proteins, spiders are able to construct silk fibers that vary tremendously in their mechanical properties, from major ampullate silk with a tensile strength rivaling that of steel to flagelliform silk with a stretchiness approaching that of rubber (Gosline et al. 1986). Despite this immense variation in physical properties and the potential exploitation of spider silks by industry, the material properties of most spider silks have never been investigated. This is due in part to the difficulty of working with silk fibers that are only a few mm in diameter at their largest, with some fibers as thin as 50–100 nm (Foelix 1996). Characterization of the mechanical properties of spider silks typically begins with measurement of the stress generated as fibers are extended until breaking (Denny 1976). Because stress is a measurement of force/cross-sectional area of a fiber, accurate and precise assessment of the diameters of fine silk threads is necessary. One of two different strategies is usually employed. The first approach is to use scanning electron microscopy (SEM) of a subsample of ‘‘focal’’ fibers that are assumed to be identical to the fibers that are to be tested mechanically. This method is expected to yield highly precise measurements of the focal fibers, but then assumes that the diameters of those focal fibers provide an accurate estimation of the diameters of the fibers that are actually tested. This could be particularly problematic when working with silks that have highly irregular diameters, such as silk collected from native structures like webs or egg sacs. The alternative strategy is to measure the diameters of each thread using a nondestructive method, such as compound light microscopy, and then to apply those diameters to each thread as they are tested mechanically. While the technical difficulties of using this procedure on fibers whose diameters approach longer wavelengths of light may decrease precision, this is the only method that can account for variation in fiber diameter within the population of samples to be tested, potentially Invertebrate Biology 124(2): 165–173. r 2005 American Microscopical Society, Inc. DOI: 10.1111/j.1744-7410.2005.00016.x Author for correspondence. Present address: Department of Biology, University of Akron, Akron, Ohio 44325-