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Proceedings Paper

Myosin rods are a source of second harmonic generation signals in skeletal muscle
Author(s): Sebastian Schürmann; Cornelia Weber; Rainer H. A. Fink; Martin Vogel
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Paper Abstract

Intrinsic second harmonic generation (SHG) signals can be used to visualize the three-dimensional structure of cardiac and skeletal muscle with high spatial resolution. Fluorescence labeling of complementary sarcomeric proteins, e.g. actin, indicates that the observed SHG signals arise from the myosin filaments. Recently, the myosin rod domain or LMM - light meromyosin - has been reported to be the dominant source of this SHG signal. However, to date, mostly negative and indirect evidence has been presented to support this assumption. Here, we show, to our knowledge, the first direct evidences that strong SHG signals can be obtained from synthetic paracrystals. These rod shaped filaments are formed from purified LMM. SDS-PAGE protein analysis confirmed that the LMM crystals lack myosin head domains. Some regions of the LMM paracrystals produce a strong SHG signal whereas others did not. The SHG signals were recorded with a laser-scanning microscope (Leica SP2). A ps laser tuned to 880 nm was used to excite the sample through an 63x objective of 1.2 NA. In order to visualize the synthetic filaments - in addition to SHG imaging -, the LMM was labeled with the fluorescent marker 5-IAF. We were able to observe filaments of 1 to 50 μm in length and of up to 5 μm in diameter. In conclusion, we can show that the myosin rod domain (LMM) is a dominant source for intrinsic SHG signals. There seems, however, a signal dependence on the paracrystals' morphology. This dependence is being investigated.

Paper Details

Date Published: 12 February 2007
PDF: 6 pages
Proc. SPIE 6442, Multiphoton Microscopy in the Biomedical Sciences VII, 64421U (12 February 2007); doi: 10.1117/12.700917
Show Author Affiliations
Sebastian Schürmann, Univ. Heidelberg (Germany)
Cornelia Weber, Univ. Heidelberg (Germany)
Rainer H. A. Fink, Univ. Heidelberg (Germany)
Martin Vogel, Ruprecht-Karls-Univ. Heidelberg (Germany)
Harvard Univ. (United States)

Published in SPIE Proceedings Vol. 6442:
Multiphoton Microscopy in the Biomedical Sciences VII
Ammasi Periasamy; Peter T. C. So, Editor(s)

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