Image correlation spectroscopy of multiphoton images correlates with collagen mechanical properties

¹ University of California, Irvine
² Norwegian University of Science and Technology

^* To whom correspondence should be addressed. E-mail: scgeorge{at}uci.edu.

Submitted on August 16, 2007
Revised on October 9, 2007
Accepted on 6 November 2007

	Abstract

Multiphoton microscopy (MPM) holds promise as a non-invasive imaging technique for characterizing collagen structure, and thus mechanical properties, through imaging second harmonic generation (SHG) and two-photon fluorescence in engineered and real connective tissues. Controlling polymerization pH to manipulate collagen gel microstructure, we quantifed pore and fiber dimensions using both standard methods and image correlation spectroscopy (ICS) on MPM, scanning electron, and darkfield microscopy images. The latter two techniques are used to confirm microstructural measurements made from MPM images. As polymerization pH increased from 5.5 to 8.5, mean fiber diameter decreased from 3.7 ± 0.7 µm to 1.6 ± 0.3 µm, the average pore size decreased from 81.7±3.7 µm² to 7.8±0.4 µm², and pore area fraction decreased from 56.8±0.8% to 18.0±1.3% (measured from SHG images), while the storage modulus G^' and the loss modulus G^'', components of the shear modulus, increased ~33-fold and ~16-fold, respectively. A characteristic length scale measured using ICS, W_ICS, correlates well with mean fiber diameter from SHG images (R² = 0.95). Semiflexible network theory predicts a scaling relationship of the collagen gel storage modulus G^' depending upon mesh size and fiber diameter, which are estimated from SHG images using ICS. We conclude that MPM and ICS are an effective combination to assess bulk mechanical properties of collagen hydrogels in a non-invasive, objective, and systematic fashion, and may be useful for specific in vivo applications.

Key Words: hydrogel, microstructure, rheometry, second harmonic, semiflexible network, two-photon fluorescence