Image correlation spectroscopy of multiphoton images correlates with collagen mechanical properties - PubMed (original) (raw)

Image correlation spectroscopy of multiphoton images correlates with collagen mechanical properties

Christopher B Raub et al. Biophys J. 2008.

Abstract

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.

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Figures

FIGURE 1

FIGURE 1

(a) Representative SHG (left column, blue) and TPF (middle column, green) images from collagen hydrogels polymerized at pH 5.5, 6.5, 7.5, and 8.5. Images are 512 × 512 pixels and 230 × 230 _μ_m. n = 12 images per condition were used for microstructure quantification and statistics. Bar represents 50 _μ_m. (b) pH-dependent fiber diameter frequency distributions were constructed from n = 358, 605, 564, and 492 fiber diameter measurements for the pH 5.5, 6.5, 7.5, and 8.5 conditions, respectively. (c) Mean pore size, (d) pore area fraction, and (e) pore density were quantified for each polymerization condition from noise thresholded, inverted SHG, or merged (SHG + TPF) images. Symbols (* and #) indicate statistical significance among polymerization pH groups.

FIGURE 2

FIGURE 2

(a) Representative SEM images reveal collagen network and fiber characteristics at 20,000× magnification (left column; bar represents 2 _μ_m) and 100,000× magnification (right column; bar represents 500 nm) for the polymerization pH conditions. n = 9, 9, 9, and 6 20,000× images were used for microstructure quantification and statistics. (b) pH-dependent fiber diameter frequency distributions were constructed from n = 477, 513, 629, and 905 fiber diameter measurements for the pH 5.5, 6.5, 7.5, and 8.5 conditions, respectively. (c) Mean pore size, pore area fraction, and pore density were quantified for each polymerization condition from noise-thresholded, inverted SEM images. Symbols (*, #, and §) indicate statistical significance among polymerization pH groups.

FIGURE 3

FIGURE 3

(a) Lengths and diameters of isolated collagen fibers were quantified using ImageJ's line-drawing feature from darkfield images of homogenized collagen hydrogels polymerized at pH 5.5 (•), 6.5 (▵), 7.5 (+), and 8.5 (▪). Each point on the graph represents the length and diameter of one fiber. n = 108 fibers were measured from each polymerization condition. Lines represent linear best fits to the data from each polymerization condition. (b) Mean fiber aspect ratio measured from darkfield images for each pH condition. Error bars represent standard deviation. Symbol (*) indicates statistical significance among pH conditions.

FIGURE 4

FIGURE 4

(a) Percentage of colocalizing SHG and TPF pixels, summed from n = 12 paired, noise-subtracted SHG and TPF images per polymerization condition with respect to total SHG and TPF signal-containing pixels. (b) Mask of a representative thresholded TPF images, showing outlines of particles with circularity 0–0.1. (c) Mask of representative thresholded TPF images, showing outlines of particles with circularity 0.5–1. (d) After applying the previous two masks separately to the noise-subtracted TPF image or its SHG counterpart, the mean signal within elliptical particles of circularity 0–0.1 (_SHG_e, _TPF_e) was divided by the mean signal within particles of circularity 0.5–1 (_SHG_c, _TPF_c). Error bars represent standard deviation. (e) formula image in arbitrary units, was estimated for various angles of tilt of the collagen fiber axis with respect to the laser propagation direction, δ: fibers at 10° and 63° of tilt are diagrammed.

FIGURE 5

FIGURE 5

_G_′ and _G_″ of collagen hydrogels, measured by parallel-plate rheometry, vary with polymerization pH. _G_′ (•) and _G_″ (▵ ) were collected at 5% strain and 10 s−1 oscillating shear frequency for n = 48 hydrogels polymerized at pH 5.5–9.0. Lines represent linear best fits to the data for _G_′ and for _G_″. The symbol * indicates that the best fit slopes are significantly different from each other; the symbols # and § indicate that each slope is significantly different from a slope of zero.

FIGURE 6

FIGURE 6

One 512 × 512 SHG image (a, raw) unprocessed, (b, HP) high-pass filtered, (c, LP) low-pass filtered, (d, NS) noise-subtracted, and (e, Th) thresholded was analyzed with ICS, and (f–j) the ACF, Gaussian fit, and residuals plotted, with the _χ_2 value of the fit reported above the residuals. (k) _W_ICS from each of the Gaussian fits.

FIGURE 7

FIGURE 7

(a) SEM images were analyzed with ICS, and _W_ICS compared to the geometric mean of hand-measured fiber diameters, d_SEM. x axis error bars represent the 1_σ lower and upper bounds around the geometric mean. (b) Thresholded (Th), noise-subtracted (NS), and unprocessed (RAW) SHG images were analyzed with ICS and by hand measurements; _d_SHG represents the arithmetic mean of hand-measured fiber diameters from SHG images. (c) _W_ICS was calculated as a function of threshold value for SHG images of collagen polymerized at the four pH values. (d) Thresholded, inverted SEM and (e) SHG images were analyzed with ICS, and _P_ICS compared to mean pore size from particle analysis, _P_PA.

FIGURE 8

FIGURE 8

Scaling relationship for the storage modulus, _G_′, was calculated using rheometric data and ICS, particle analysis, or hand-measured estimates of mesh size and fiber diameter from SHG images. Best-fit slopes and _R_2 values are given in the figure.

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