Studies of χ(2)/χ(3) Tensors in Submicron-Scaled Bio-Tissues by Polarization Harmonics Optical Microscopy

Abstract

Optical second- and third-harmonic generations have attracted a lot of attention in the biomedical imaging research field recently due to their intrinsic sectioning ability and noninvasiveness. Combined with near-infrared excitation sources, their deep-penetration ability makes these imaging modalities suitable for tissue characterization. In this article, we demonstrate a polarization harmonics optical microscopy, or P-HOM, to study the nonlinear optical anisotropy of the nanometer-scaled myosin and actin filaments inside myofibrils. By using tight focusing we can avoid the phase-matching condition due to micron-scaled, high-order structures in skeletal muscle fibers, and obtain the submicron-scaled polarization dependencies of second/third-harmonic generation intensities on the inclination angle between the long axes of the filaments and the polarization direction of the linear polarized fundamental excitation laser light. From these dependencies, detailed information on the tensor elements of the second/third-order nonlinear susceptibilities contributed from the myosin/actin filaments inside myofibrils can thus be analyzed and obtained, reflecting the detailed arrangements and structures of the constructing biomolecules. By acquiring a whole, nonlinearly sectioned image with a submicron spatial resolution, we can also compare the polarization dependency and calculate the nonlinear susceptibilities over a large area of the tissue at the same time-which not only provides statistical information but will be especially useful with complex specimen geometry.

Imaging the bipolarity of myosin filaments with Interferometric Second Harmonic Generation microscopy

“…While rotating the polarization of the pump laser to maximize SHG in the muscle tissue, we observed the same dependence on the SHG intensity as the one reported by Chu (Fig. 4(a) [13]), Plotnikov ( Fig. 6(b) [6]) and Tiaho ( Fig.…”

“…Standard SHG microscopy can easily be performed with the same setup after removing or rotating by 90° the reference quartz plate. Myosin filaments have non-zero χ zxx , χ xzx and χ zzz in their χ (2) tensor because of their C ∞ symmetry [13,14]. While rotating the polarization of the pump laser to maximize SHG in the muscle tissue, we observed the same dependence on the SHG intensity as the one reported by Chu (Fig.…”

“…The dependence of the SHG intensity on the pump laser polarization is explained by the weakness of χ zzz in muscle compared to χ zxx and χ xzx . Note that ISHG microscopy is sensitive to these tensor elements since they are achiral in nature [13,36]. The polarization of the laser and the analyzer were oriented to maximize the yield of SHG signal at the photomultiplier tube.…”

“…Since the myosin filaments have a noncentrosymmetric structural organization, muscle tissues can be imaged using Second Harmonic Generation (SHG) microscopy [6][7][8][9][10][11][12][13][14][15][16]. Other biological structures possess a noncentrosymmetric organization such as tissues rich in collagen type I/III proteins [14][15][16][17][18][19][20][21][22], collagen type 2 [22][23][24] and the microtubules within cells [25].…”

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). A particularly strong attribute driving these efforts is that the SHG contrast is produced purely from the endogenous proteins without the need for exogenous dyes.…”