Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence
Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence
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DOI：
10.1016/j.jsb.2008.03.010
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发表时间：
2008-06-01
影响因子：
3
通讯作者：
Raben, N.
中科院分区：
文献类型：
--
作者：
Ralston, E.;Swaim, B.;Raben, N.
骨骼肌的多核肌纤维的大尺寸使得它们的结构和病理缺陷的检查成为挑战。切片和单纤维可以接触到抗体和其他标记物，但这些样品的成像不能提供肌肉的三维视图。遗憾的是，纤维束不能容易地染色或成像。双光子显微镜技术克服了这些障碍。二次谐波产生（SHG）的肌球蛋白丝和双光子激发荧光（2PEF）的线粒体和溶酶体成分提供了详细的结构信息未染色的组织。此外，红外激发光可以穿透几层肌肉纤维，并且最小的处理对于脆弱的活检特别有价值。在这里，我们证明了SHG的有用性，结合2PEF，揭示扩大的溶酶体和积累的非收缩性材料的肌肉从小鼠模型的溶酶体贮积症庞贝氏症（PD），并在活检从成人和婴儿PD患者。SHG和2PEF还检测肌节缺陷，这可能预示着萎缩肌肉中肌原纤维的损失，并表示弹性的损失。SHG和2PEF的组合应该是有用的，在分析和诊断范围广泛的骨骼肌病变。爱思唯尔公司出版
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. Published by Elsevier Inc.