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Noninvasive Assessment of Collagen Gel Microstructure and Mechanics Using Multiphoton Microscopy

Christopher B. Raub ^*, Vinod Suresh ^*, Tatiana Krasieva , Julia Lyubovitsky , Justin D. Mih ^*, Andrew J. Putnam ^* , Bruce J. Tromberg ^*   and Steven C. George ^* 

^* Department of Biomedical Engineering, The Beckman Laser Institute, Department of Chemical Engineering and Materials Science, and Department of Surgery, University of California Irvine, Irvine, California

Correspondence: Address reprint requests to Steven C. George, MD, PhD, Dept. of Biomedical Engineering, 3120 Natural Sciences II, University of California Irvine, Irvine, CA 92697-2715. Tel.: 949-824-3941; Fax: 949-824-2541; E-mail: scgeorge{at}uci.edu.

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 (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°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°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.

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