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.

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Multiphoton microscopy (MPM) holds promise as a noninvasive 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 quantified 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 microm to 1.6 +/- 0.3 microm, the average pore size decreased from 81.7 +/- 3.7 microm(2) to 7.8 +/- 0.4 microm(2), and the pore area fraction decreased from 56.8% +/- 0.8% to 18.0% +/- 1.3% (measured from SHG images), whereas the storage modulus G' and loss modulus G'', components of the shear modulus, increased approximately 33-fold and approximately 16-fold, respectively. A characteristic length scale measured using ICS, W(ICS), correlates well with the mean fiber diameter from SHG images (R(2) = 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 noninvasive, objective, and systematic fashion and may be useful for specific in vivo applications.

Cellularized collagen gels are a common model in tissue engineering, but the relationship between the microstructure and bulk mechanical properties is only partially understood. Multiphoton microscopy (MPM) is an ideal non-invasive tool to examine collagen microstructure, cellularity and crosslink content in these gels. In order to identify robust image parameters that characterize microstructural determinants of the bulk elastic modulus, we performed serial MPM and mechanical tests on acellular and cellularized (normal human lung fibroblasts) collagen hydrogels, before and after glutaraldehyde crosslinking. Following gel contraction over sixteen days, cellularized collagen gel content approached that of native connective tissues (~200 mg/ml). Young’s modulus (E) measurements from acellular collagen gels (range 0.5-12 kPa) exhibited a power-law concentration dependence (range 3-9 mg/ml) with exponents from 2.1-2.2, similar to other semiflexible biopolymer networks such as fibrin and actin. In contrast, cellularized collagen gel stiffness (range 0.5-27 kPa) produced concentration-dependent exponents of 0.7 uncrosslinked and 1.1 crosslinked (range ~5-200 mg/ml). The variation in E of cellularized collagen hydrogels can be explained by a power-law dependence on robust image parameters: either the second harmonic generation (SHG) and two-photon fluorescence (TPF) (matrix component) skewness (R2 = 0.75, exponents of −1.0 and −0.6, respectively); or alternately the SHG and TPF (matrix component) speckle contrast (R2 = 0.83, exponents of −0.7 and −1.8, respectively). Image parameters based on the cellular component of TPF signal did not improve the fits. The concentration dependence of E suggests enhanced stress relaxation in cellularized versus acellular gels. SHG and TPF image skewness and speckle contrast from cellularized collagen gels can predict E by capturing mechanically relevant information on collagen fiber, cell and crosslink density.