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Progressive force loss in Duchenne muscular dystrophy is characterized by degeneration/regeneration cycles and fibrosis. Disease progression may involve structural remodeling of muscle tissue. An effect on molecular motorprotein function may also be possible. We used second harmonic generation imaging to reveal vastly altered subcellular sarcomere microarchitecture in intact single dystrophic mdx muscle cells (approximately 1 year old). Myofibril tilting, twisting, and local axis deviations explain at least up to 20% of force drop during unsynchronized contractile activation as judged from cosine angle sums of myofibril orientations within mdx fibers. In contrast, in vitro motility assays showed unaltered sliding velocities of single mdx fiber myosin extracts. Closer quantification of the microarchitecture revealed that dystrophic fibers had significantly more Y-shaped sarcomere irregularities ("verniers") than wild-type fibers (approximately 130/1000 microm(3) vs. approximately 36/1000 microm(3)). In transgenic mini-dystrophin-expressing fibers, ultrastructure was restored (approximately 38/1000 microm(3) counts). We suggest that in aged dystrophic toe muscle, progressive force loss is reflected by a vastly deranged micromorphology that prevents a coordinated and aligned contraction. Second harmonic generation imaging may soon be available in routine clinical diagnostics, and in this work we provide valuable imaging tools to track and quantify ultrastructural worsening in Duchenne muscular dystrophy, and to judge the beneficial effects of possible drug or gene therapies.

The pore size of biopolymer networks governs their mechanical properties and strongly impacts the behavior of embedded cells. Confocal reflection microscopy and second harmonic generation microscopy are widely used to image biopolymer networks; however, both techniques fail to resolve vertically oriented fibers. Here, we describe how such directionally biased data can be used to estimate the network pore size. We first determine the distribution of distances from random points in the fluid phase to the nearest fiber. This distribution follows a Rayleigh distribution, regardless of isotropy and data bias, and is fully described by a single parameter--the characteristic pore size of the network. The bias of the pore size estimate due to the missing fibers can be corrected by multiplication with the square root of the visible network fraction. We experimentally verify the validity of this approach by comparing our estimates with data obtained using confocal fluorescence microscopy, which represents the full structure of the network. As an important application, we investigate the pore size dependence of collagen and fibrin networks on protein concentration. We find that the pore size decreases with the square root of the concentration, consistent with a total fiber length that scales linearly with concentration.

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Skeletal muscle is an archetypal organ whose structure is tuned to match function. The magnitude of order in muscle fibers and myofibrils containing motor protein polymers determines the directed force output of the summed force vectors and, therefore, the muscle’s power performance on the structural level. Structure and function can change dramatically during disease states involving chronic remodeling. Cellular remodeling of the cytoarchitecture has been pursued using noninvasive and label-free multiphoton second harmonic generation (SHG) microscopy. Hereby, structure parameters can be extracted as a measure of myofibrillar order and thus are suggestive of the force output that a remodeled structure can still achieve. However, to date, the parameters have only been an indirect measure, and a precise calibration of optical SHG assessment for an exerted force has been elusive as no technology in existence correlates these factors.  We engineered a novel, automated, high-precision biomechatronics system into a multiphoton microscope allows simultaneous isometric Ca2+-graded force or passive viscoelasticity measurements and SHG recordings. Using this MechaMorph system, we studied force and SHG in single EDL muscle fibers from wt and mdx mice; the latter serves as a model for compromised force and abnormal myofibrillar structure. We present Ca2+-graded isometric force, pCa-force curves, passive viscoelastic parameters and 3D structure in the same fiber for the first time. Furthermore, we provide a direct calibration of isometric force to morphology, which allows noninvasive prediction of the force output of single fibers from only multiphoton images, suggesting a potential application in the diagnosis of myopathies.

This review focuses on modern nonlinear optical microscopy (NLOM) methods that are increasingly being used in the field of tissue engineering (TE) to image tissue non-invasively and without labelling in depths unreached by conventional microscopy techniques. With NLOM techniques, biomaterial matrices, cultured cells and their produced extracellular matrix may be visualized with high resolution. After introducing classical imaging methodologies such as µCT, MRI, optical coherence tomography, electron microscopy and conventional microscopy two-photon fluorescence (2-PF) and second harmonic generation (SHG) imaging are described in detail (principle, power, limitations) together with their most widely used TE applications. Besides our own cell encapsulation, cell printing and collagen scaffolding systems and their NLOM imaging the most current research articles will be reviewed. These cover imaging of autofluorescence and fluorescence-labelled tissue and biomaterial structures, SHG-based quantitative morphometry of collagen I and other proteins, imaging of vascularization and online monitoring techniques in TE. Finally, some insight is given into state-of-the-art three-photon-based imaging methods (e.g. coherent anti-Stokes Raman scattering, third harmonic generation). This review provides an overview of the powerful and constantly evolving field of multiphoton microscopy, which is a powerful and indispensable tool for the development of artificial tissues in regenerative medicine and which is likely to gain importance also as a means for general diagnostic medical imaging.

The second harmonic generation (SHG) signal intensity sourced from skeletal muscle myosin II strongly depends on the polarization of the incident laser beam relative to the muscle fiber axis. This dependence is related to the second-order susceptibility χ((2)), which can be described by a single component ratio γ under generally assumed symmetries. We precisely extracted γ from SHG polarization dependence curves with an extended focal field model. In murine myofibrillar preparations, we have found two distinct polarization dependencies: With the actomyosin system in the rigor state, γ(rig) has a mean value of γ(rig) = 0.52 (SD = 0.04, n = 55); in a relaxed state where myosin is not bound to actin, γ(rel) has a mean value of γ(rel) = 0.24 (SD = 0.07, n = 70). We observed a similar value in an activated state where the myosin power stroke was pharmacologically inhibited using N-benzyl-p-toluene sulfonamide. In summary, different actomyosin states can be visualized noninvasively with SHG microscopy. Specifically, SHG even allows us to distinguish different actin-bound states of myosin II using γ as a parameter.

In striated muscle, desmin intermediate filaments interlink the contractile myofibrillar apparatus with mitochondria, nuclei, and the sarcolemma. The desmin network’s pivotal role in myocytes is evident since mutations in the human desmin gene cause severe myopathies and cardiomyopathies. Here, we investigated skeletal muscle pathology in myofibers and myofibrils isolated from young hetero- and homozygous R349P desmin knock-in mice, which carry the orthologue of the most frequent human desmin missense mutation R350P. We demonstrate that mutant desmin alters myofibrillar cytoarchitecture, markedly disrupts the lateral sarcomere lattice and distorts myofibrillar angular axial orientation. Biomechanical assessment revealed a high predisposition to stretch-induced damage in fiber bundles of R349P mice. Notably, Ca2 +-sensitivity and passive myofibrillar tension were decreased in heterozygous fiber bundles, but increased in homozygous fiber bundles compared to wildtype mice. In a parallel approach, we generated and subsequently subjected immortalized heterozygous R349P desmin knock-in myoblasts to magnetic tweezer experiments that revealed a significantly increased sarcolemmal lateral stiffness. Our data suggest that mutated desmin already markedly impedes myocyte structure and function at pre-symptomatic stages of myofibrillar myopathies.