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.

Phase matching considerations in second harmonic generation from tissues: Effects on emission directionality, conversion efficiency and observed morphology

“…To analyze the incoherent scattering component, we performed Monte Carlo simulations of the photon propagation based on our measured bulk optical properties at both the fundamental and SHG wavelengths. (19) Simulations assuming 100% forward generation reproduced the measured trends but overestimated the detected F/B at all depths. Based on previous reports, (4,18) this is not a realistic scenario since for small fibrils a significant backward coherent component will exist.…”

“…Differentiating these components is an important consideration for clinical applications as diseased states may have smaller and more randomly packed fibrils which affect the initial generation directionality. Such tissues may also have different bulk optical parameters, governing the subsequent photon propagation (19).…”

“…We have recently examined the mixing of the quasi-coherent and incoherent contributions in fibrillar cellulose (18) as well as in OI and normal murine skin (19) and found the relative component weights are highly dependent upon SHG creation depth. Experimentally, we measured the SHG forward to backward ratio (F/B) which results from both components, as a function of the excitation depth into the tissue.…”

From molecular structure to tissue architecture: collagen organization probed by SHG microscopy

“…As depicted in Figure 4, the signals detected in forward and backward directions drastically depend on the sample observed: while in cornea and tendon (Figure 4a) the signal is mainly directed in forward direction, in sclera and skin (Figure 4b) there is a relevant back-scattered component. An example of application to tissue disease is represented in Figure 4c, d, where the measurement of the forwardbackward SHG ratio allowed to discriminate a normal tissue sample against a sample affected by osteogenesis imperfecta (WT and OIM in Figure 4d, respectively) [96]. [66] and tendon [106]) and by a comparable amount of forward-backward-scattered signal (b) -sclera [66] and skin).…”

“…In fact, SHG microscopy highlights morphologic changes in collagen structure, which indicate particular disease states, such as tumour invasiveness, as well as indicators of collagen remodelling in tumour stroma, which is playing a key-role in the tumour development from in-situ to invasive stage. Recently, SHG microscopy has been successfully applied to the study of altered physiological conditions of various kinds of tissues, including muscle [94], bones [95,96], and cartilages [97,98]. SHG can be easily combined with TPEF microscopy to realize a particularly powerful tool for connective tissue imaging.…”