Investigating The Network Structure Of Type I Collagen As A Function Of Temperature And Concentration Via Confocal Microscopy

Abstract

Three dimensional in vitro approximations to extracellular matrix (ECM) are increasingly being used in biophysical experiments investigating cell behavior. One advantage to using collagen I gels as ECM approximations in such experiments is the ability to image the collagen fibers within the gel. This allows simultaneous imaging of cells and their local environment simultaneously with three dimensional (3D) resolution. In this study, both pure and fluorescently labeled (3% by weight) collagen gels are assembled over a range of concentrations (0.1, 0.5, 1.0, 2.0, 5.0, and 5.5 mg/mL) and at various temperatures (37, 32, 27, and 22°C). The networks are investigated using confocal reflectance microscopy (CRM) and confocal fluorescence microscopy (CFM). Comparison between CRM and CFM reveals that they are not equally sensitive to details of network structure, with CRM (CFM) displaying higher sensitivity to fibers perpendicular (parallel) to the optical axis. Furthermore, analysis of background signal in CFM images suggests the existence of small fibrillar structures that are not resolved by CRM. Despite these differences, image analyses performed on 2D slices of CFM and CRM images to quantify mesh size, number of fibers, and fiber length reveal identical trends as a function of gel concentration and gelation temperature. Fiber width approximated from both CRM and CFM is in good accord with fiber width determination using electron microscopy. Overall network structures (as quantified via mesh size, fiber number, fiber length and fiber width) are related to bulk mechanical properties measured by rheology. Finally, we demonstrate the ability to form collagen gels of varied mesh size at identical collagen concentration, all of which are compatible with cell health and 3D cell studies.