title: Noninvasive assessment of collagen gel microstructure and mechanics using multiphoton microscopy.
abstract: Multiphoton microscopy of collagen hydrogels produces second harmonic generation (SHG) and two-photon fluorescence (TPF) images, which can be used to noninvasively study gel microstructure at depth ( approximately 1 mm). The microstructure is also a primary determinate of the mechanical properties of the gel; thus, we hypothesized that bulk optical properties (i.e., SHG and TPF) could be used to predict bulk mechanical properties of collagen hydrogels. We utilized polymerization temperature (4-37 degrees C) and glutaraldehyde to manipulate collagen hydrogel fiber diameter, space-filling properties, and cross-link density. Multiphoton microscopy and scanning electron microscopy reveal that as polymerization temperature decreases (37-4 degrees C) fiber diameter and pore size increase, whereas hydrogel storage modulus (G', from 23 +/- 3 Pa to 0.28 +/- 0.16 Pa, respectively, mean +/- SE) and mean SHG decrease (minimal change in TPF). In contrast, glutaraldehyde significantly increases the mean TPF signal (without impacting the SHG signal) and the storage modulus (16 +/- 3.5 Pa before to 138 +/- 40 Pa after cross-linking, mean +/- SD). We conclude that SHG and TPF can characterize differential microscopic features of the collagen hydrogel that are strongly correlated with bulk mechanical properties. Thus, optical imaging may be a useful noninvasive tool to assess tissue mechanics.
authors.affiliation: Department of Biomedical Engineering, University of California Irvine, Irvine, California 92697-2715, USA.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Raub
authors.forename: Vinod
authors.initials: V
authors.lastname: Suresh
authors.forename: Tatiana
authors.initials: T
authors.lastname: Krasieva
authors.forename: Julia
authors.initials: J
authors.lastname: Lyubovitsky
authors.forename: Justin D
authors.initials: JD
authors.lastname: Mih
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Putnam
authors.forename: Bruce J
authors.initials: BJ
authors.lastname: Tromberg
authors.forename: Steven C
authors.initials: SC
authors.lastname: George
mesh_terms: D001696:Biomechanical Phenomena / Q000379:methods; D024042:Collagen Type I / Q000737:chemistry* / Q000648:ultrastructure*; D004548:Elasticity; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D008968:Molecular Conformation; D010316:Particle Size; D013314:Stress, Mechanical
chemical_list: D024042:Collagen Type I