Collagen fibril diameter quantification using interference confocal reflectance microscopy

Understanding collagen fibril assembly and remodeling is critical to understanding the kinetics of assembling larger collagen structures. Being able to quantify a fibril's mechanics while interacting with the collagen molecule would be a large step towards understanding how fibrils both self-assemble and remodel with larger collagen structures. In-vitro experimentation with individual collagen fibrils has opened the door to many possibilities for understanding collagen at the nanometer scale; however, many collagen fibrils have diameters that lie below the Abbe diffraction limit and therefore cannot be quantified with traditional microscopy methods. This presents a barrier for being able to quantify a fibril's mechanical properties (stress, toughness) while simultaneously interacting with collagen monomers. The objective of this study was to design a collagen fibril diameter measurement method using confocal reflectance microscopy that could quantify fibril diameters below the diffraction limit, ideally well below 300 nm. In addition, the method should be able to integrate with confocal fluorescence systems, which are used to track collagen monomers and labeled collagen fibrils. This setup would allow real-time quantification of how collagen monomers interact with existing fibrils to grow and repair over time, while under cyclic loading. A 2D finite difference time domain (FDTD) simulation was first used to estimate the interference patterns at each excitation wavelength. Type 1 collagen fibrils (dry) were stretched over polydimethylsiloxane (PDMS) trenches, ∼25 µm wide, and imaged using a Zeiss LSM 880 confocal reflectance microscope. The selected wavelengths were 488 nm, 514 nm, and 561 nm. Fibrils were then imaged using a Hitachi S-4800 FE-SEM to estimate the range of fibrils imaged and used for assisting algorithm development. The interference pattern from the confocal reflectance imaging results matched the expected shape found in the 2D FDTD. These results were then fed through an algorithm developed with the FDTD to estimate the collagen fibril's diameter, and directly overlaid with the registered scanning electron microscopy (SEM) images. Interference confocal reflectance microscopy (I-CRM) provides a promising modality for measuring collagen fibril diameters well below the diffraction limit. It provides several advantages over other methods, including non-lethality, no fluorescent staining, and a particularly promising ability for no-registration calibration.