Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence

Abstract

The large size of the multinucleated muscle fibers of skeletal muscle makes their examination for structural and pathological defects a challenge. Sections and single fibers are accessible to antibodies and other markers but imaging of such samples does not provide a three-dimensional view of the muscle. Regrettably, bundles of fibers cannot be stained or imaged easily. Two-photon microscopy techniques overcome these obstacles. Second harmonic generation (SHG) by myosin filaments and two-photon excited fluorescence (2PEF) of mitochondrial and lysosomal components provides detailed structural information on unstained tissue. Furthermore, the infrared exciting light can penetrate several layers of muscle fibers and the minimal processing is particularly valuable for fragile biopsies. Here we demonstrate the usefulness of SHG, combined with 2PEF, to reveal enlarged lysosomes and accumulations of non-contractile material in muscles from the mouse model for the lysosomal storage disorder Pompe disease (PD), and in biopsies from adult and infant PD patients. SHG and 2PEF also detect sarcomeric defects that may presage the loss of myofibrils in atrophying muscle and signify loss of elasticity. The combination of SHG and 2PEF should be useful in the analysis and diagnosis of a wide range of skeletal muscle pathologies.

Introduction

Muscle pathologies are traditionally investigated and diagnosed by light microscopy of frozen or embedded tissue sections, stained with histochemical markers or antibodies (Engel and Franzini-Armstrong, 2004). For high resolution, electron microscopy produces unparalleled detail quality (ibidem). However, these methods produce an image of a thin slice of tissue and the reconstruction of a three-dimensional view requires painstaking serial sectioning and/or good knowledge of stereometrics. Single whole muscle fibers, stained for immunofluorescence, can be scanned in the confocal microscope to provide a complete three-dimensional view of the individual fiber (Ralston and Ploug, 1996, Ploug et al., 1998). However, the context of the larger muscle organization is lost once the fibers are teased from one another. Furthermore, it is difficult to ensure that all fiber types and conditions are represented when the pathology affects some of them more than others. The ideal technique would allow high-resolution imaging of fiber bundles with minimal need for processing. Two-photon (2P) microscopy techniques such as SHG bring us close to this goal. SHG is a non-linear technique in which two photons recombine in a non-resonant process to produce one photon of half the energy when non-centro-symmetric molecular arrays are present (Campagnola and Loew, 2003). Collagen, in the basal lamina which surrounds muscle fibers, and myosin thick filaments, in the core of the fibers, are emitters of SHG (Both et al., 2004, Plotnikov et al., 2006a, Plotnikov et al., 2006b, Greenhalgh et al., 2007). The absence of energy absorption in this process permits deep penetration with minimal tissue damage and little signal loss (Mohler et al., 2003); relatively large bundles of unstained muscle fibers can be examined throughout. In addition, the intensity of the SHG signal is exquisitely sensitive to the orientation of the emitting molecules (Vanzi et al., 2006) and to the semi-crystalline order of the myofibrils (Greenhalgh et al., 2007). Skeletal muscle is also rich in intrinsically fluorescent components (Zipfel et al., 2003, Rothstein et al., 2005) such as flavins (FMN and FAD) and NAD(P)H in mitochondria as well as the intralysosomal pigment lipofuscin. Lipofuscin, an indigestible material which is formed by cross-linking of protein residues accumulates due to age and oxidative damage (Brunk and Terman, 2002, Hutter et al., 2007).

Proof-of-principle papers have given detailed information on intrinsically fluorescent and SHG-emitting molecules in various tissues and have shown that the two techniques can be usefully combined in vitro (Zoumi et al., 2002, Zipfel et al., 2003) and in vivo (Rothstein et al., 2006). SHG, alone or with 2PEF, has also been used to image collagen in the cornea (Yeh et al., 2002, Han et al., 2004), cartilage (Yeh et al., 2005), skin (Chen et al., 2006, Lin et al., 2006), and dentin (Elbaum et al., 2007), as well as in mouse tumors in vivo (Brown et al., 2003) and in a mouse model of osteogenesis imperfecta (Nadiarnykh et al., 2007). These studies have amply demonstrated that SHG should be useful to study conditions that affect connective tissue. Muscle applications, however, have lagged behind, perhaps because myosin is a weaker SHG emitter than collagen.

We are interested in Pompe disease (PD), an inherited lysosomal storage disorder resulting from mutations in acid α-glucosidase (GAA; also called acid maltase) (Hirschhorn and Reuser, 2001, Futerman and van Meer, 2004). Although the defect is present in all tissues, the pathology is most detrimental to skeletal and cardiac muscle. The telltale sign of the disease is the accumulation of glycogen in enlarged lysosomes, but when we stained single muscle fibers of GAA KO mice, the mouse model of PD (Raben et al., 1998), we also noticed large inclusions of autophagic debris in every type II (fast-twitch) fiber (Fukuda et al., 2006). These inclusions interrupt the myofibrils and could interfere with muscle contraction (Drost et al., 2005). The autophagic component is also present in muscle fibers from Pompe patients (Engel, 1970, Raben et al., 2007). However, large variability between patients and between fibers within a single biopsy makes the quantitation of these areas difficult. In addition, the recent approval of enzyme replacement therapy for PD and the emergence of newborn screening programs call for tools allowing complete scanning of valuable biopsies and high sensitivity in the detection of muscle defects.

We now demonstrate that the combination of the 2P imaging techniques SHG, detected in the forward direction (transmitted), and excited fluorescence (2PEF), recorded in backscattered mode, detects the whole range of muscle defects in PD, some of which were previously inaccessible. An additional benefit of this approach is the considerable simplification of sample preparation.