Quantitative second harmonic generation imaging of the diseased state osteogenesis imperfecta: experiment and simulation

Abstract

We report the integrated use of 3D second harmonic generation (SHG) imaging microscopy and Monte Carlo simulation as a combined metric to quantifiably differentiate normal and diseased tissues based on the physical properties of the respective extracellular matrix. To achieve this, we have identified a set of parameters comprised of the SHG creation attributes and the bulk optical parameters, which are used collectively via comparative analysis. Monte Carlo simulations of the SHG axial directional and attenuation responses allow their decomposition into the underlying factors that are not readily obtainable through experimental techniques. Specifically, this approach allows for estimation of the SHG creation attributes (directionality and relative conversion efficiency) and separation of primary and secondary filter effects, collectively that form the observed SHG contrast. The quantitative metric is shown for the connective tissue disorder Osteogenesis Imperfecta (characterized by abnormal assembly of type I collagen) using a murine model that expresses the disease in the dermis layer of skin. Structural dissimilarities between the osteogenesis imperfecta mouse and wild-type tissues lead to significant differences in the SHG depth-dependent directionality and signal attenuation. The Monte Carlo simulations of these responses using measured bulk optical parameters reproduce the experimental data trends, and the extracted emission directionality and conversion efficiencies are consistent with independent determinations. The simulations also illustrate the dominance of primary filter affects on overall SHG generation and attenuation. Thus, the combined method of 3D SHG imaging and modeling forms an essential foundation for parametric description of the matrix properties that are not distinguishable by sole consideration of either bulk optical parameters or SHG alone. Moreover, due to the quasi-coherence of the SHG process in tissues, we submit that this approach contains unique information not possible by purely scattering based methods and that these methods will be applicable in the general case where the complex fibrillar structure is difficult to fully quantify via morphological analysis.

FIGURE 1

Flowchart of the algorithm used in the Monte Carlo simulations of the axial dependences of the measured SHG directionality and attenuation.

FIGURE 2

SHG images of WT (left) and oim dermis (right) dermis. (a) Individual optical sections. (b) 3D rendering of corresponding z series. The oim dermis is significantly thinner than that of the WT (∼30 vs. 60 μm). Consistent with SE data, the WT fibrils are larger and more densely packed. Scale bar = 25 μm.

FIGURE 3

Experimental data and fits to Eq. 2 for the scattering anisotropy, g, for the oim (squares) and WT (circles). These fits yielded g values of 0.68 and 0.78 for the oim and WT skin, respectively.

FIGURE 4

Ratio of forward and backward collected SHG as a function of depth into oim and WT skin. These photon propagation data are consistent with a multiple scattering process.

FIGURE 5

(a) Representative Monte Carlo simulations of the measured depth dependent directionality (F/B) for the oim and WT skin with SHG creation emission directions of F_SHG = 100% and 50%. Simulations were carried out over the range of 100–40% forward emission. Fitting these responses to the experimental data resulted in 77.5% and 72.5% F_SHG emission for the oim and WT, respectively. (b) Comparison of the Monte Carlo simulations of the oim and WT F/B assuming 77.5% and 72.5% F_SHG creation emission for the oim and WT, respectively with the experimental data. The standard error in the simulations results from the standard errors in the bulk optical parameters shown in Table 1 at the SHG wavelength. χ²- tests for both the WT and oim indicate that the respective experimental and simulated results are not significantly different.

FIGURE 6

Comparison of the experimental forward SHG attenuation data with Monte Carlo simulations (with associated SE) based on the bulk optical parameters at both the fundamental and SHG wavelengths (Table 1). The creation directionality was taken from Fig. 5b, and relative SHG conversion efficiency of 2.54-fold larger for the WT was used. As absolute magnitude of the SHG intensity from the oim is smaller than that of the WT, the data are normalized to their respective maximum and also to the maximum in each series to account for local variability in the tissues. χ²- tests for both the WT and oim indicate that the respective experimental and simulated results are not significantly different.

FIGURE 7

Comparison of Monte Carlo simulations (using the bulk optical parameters Table 1) of the primary filter effect and the measured SHG attenuation (primary + secondary filters) for the WT and oim skin. These simulations are similar in their depth-dependence showing that the attenuation response is set primarily by the primary filter.