Polarization-dependent optical second-harmonic imaging of a rat-tail tendon

Abstract

Using scanning confocal microscopy, we measure the backscattered second harmonic signal generated by a 100 fs laser in rat-tail tendon collagen. Damage to the sample is avoided by using a continuous scanning technique, rather than measuring the signal at discrete points. The second harmonic signal varies by about a factor of 2 across a single cross section of the rat-tail tendon fascicle. The signal intensity depends both on the collagen organization and the backscattering efficiency. This implies that we cannot use intensity measurements alone to characterize collagen structure. However, we can infer structural information from the polarization dependence of the second harmonic signal. Axial and transverse scans for different linear polarization angles of the input beam show that second harmonic generation (SHG) in the rat-tail tendon depends strongly on the polarization of the input laser beam. We develop an analytical model for the SHG as a function of the polarization angle in the rat-tail tendon. We apply this model in determining the orientation of collagen fibrils in the fascicle and the ratio gamma between the two independent elements of the second-order nonlinear susceptibility tensor. There is a good fit between our model and the measured data.

Fully automated, quantitative, noninvasive assessment of collagen fiber content and organization in thick collagen gels

“…Also, the confinement of SHG excitation within a small volume eliminates the need for a confocal detection pinhole and allows for more efficient signal collection. However, the efficiency of SHG generation depends on the relative orientation of the collagen fibers with respect to the polarization of the incident beam, 17,18,22,33,34 the arrangement of adjacent collagen fibrils 34 and environmental factors such as ionic strength 14 and this may introduce errors in the collagen content estimates performed by SHG image analysis. The use or circularly polarized light and three-dimensional image analysis approaches may offer options for overcoming some of these effects.…”

“…Polarizationdependent and polarization-modulated measurements could offer more accurate and detailed information on fiber organization, but they require more involved data acquisition procedures and may be more suitable for thin samples. 22,34 In summary, these studies illustrate that dynamic monitoring along with automated and easy to implement image analysis approaches could yield important information with regards to tissue status and function. These approaches will become increasingly more useful as SHG imaging finds broader use in in vivo studies for tissue engineering, wound healing, disease diagnostic, and drug assessment applications.…”

“…17,18 Finally, the degree of organization and alignment within fibrillar bundles has been found to significantly influence the detected SHG signal. 19 The analytical tools that have been presented so far for characterizing collagen organization relied at least to a certain extent either on user input, 8,12,17,[19][20][21][22] or on computationally intense calculations that confine the region of interest to a limited region. 9 Here, we present the use of an adaptive thresholding approach in combination with Fourier-and a Hough-transform based analysis as a relatively simple and efficient means of acquiring quantitative information on the content, orientation, and local organization of collagen fibrils.…”

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

“…The majority of the recent reports have focused on visualizing collagen fibers in natural tissues including skin, tendon, blood vessels, and cornea (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). A smaller body of publications has described SHG imaging of acto-myosin complexes in muscle (13,14) as well as microtubule-based structures in live cells (13,15).…”

Polarization-resolved second-harmonic generation microscopy as a method to visualize protein-crystal domains

“…PR-SHG measurements of monolayers at interfaces have previously been shown to enable discrimination between samples with similar nonlinear optical properties (Begue, Everly et al, 2009;Begue & Simpson, 2010;Begue, Moad et al, 2009). Further, polarization-dependent SHG microscopy has a rich history of enabling structural and orientational studies Amat-Roldan et al, 2010;Chang et al, 2011;Latour et al, 2012;Duboisset et al, 2012;Stoller et al, 2002;Mansfield et al, 2007;Nucciotti et al, 2009;Filippidis et al, 2009;Madden et al, 2011;Tuer et al, 2011;Brideau & Stys, 2012). PCA offers the advantages of simplicity, generality and the absence of required training when extracting the core features of high-dimensional data.…”