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Proceedings Paper

4Pi-SHG imaging of mammalian myofibrillar structures
Author(s): Martin Vogel; Dorothea Hahn; Sebastian Schürmann; Marion Lang; Frederic von Wegner; Oliver Friedrich; Johann Engelhardt; Stefan W. Hell; Rainer H. A. Fink
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Paper Abstract

Intrinsic Second Harmonic Generation (SHG) signals obtained from the motor protein myosin are of particular interest for 3D-imaging of living muscle cells. In addition, the new and powerful tool of 4Pi microscopy allows to markedly enhance the optical resolution of microscopy as well as the sensitivity for small objects because of the high peak intensities due to the interference pattern created in the focus. In the present study, we report, to our knowledge for the first time, measurements of intrinsic SHG signals under 4Pi conditions of type A. These measurements on mammalian myofibrilar structures are combined with very high resolution 4Pi fluorescence data obtained from the same preparations. We have chosen myofibrillar preparations of isolated mammalian muscle fibers as they (i) possess a regular repetitive pattern of actin and myosin filaments within sarcomers 2 to 3 μm in length, (ii) consist of single myofibrils of small total diameter of approximately 1 μm and (iii) are ideally suited to study the biomedically important process of force generation via calcium regulated motor protein interactions. Myofibrillar preparations were obtained from murine skeletal and heart muscle by using a combined chemical and mechanical fractionation1 (Both et al. 2004, JBO 9(5):882-892). BODIPY FL phallacidin has been used to fluorescently label the actin filaments. The experiments were carried out with a Leica SP2 multi photon microscope modified for 4Pi measurements using a Ti:Sa laser tuned to 850-900 nm. SHG as well as fluorescence photons were detected confocally by a counting APD detector. The approach taken our study provides new 3D-data for the analysis and simulation of the important process of excitation-contraction coupling under normal physiological as well as under pathophysiological conditions.

Paper Details

Date Published: 23 February 2006
PDF: 8 pages
Proc. SPIE 6089, Multiphoton Microscopy in the Biomedical Sciences VI, 60891Q (23 February 2006); doi: 10.1117/12.646638
Show Author Affiliations
Martin Vogel, Ruprecht-Karls-Univ. Heidelberg (Germany)
Dorothea Hahn, German Cancer Research Ctr. (Germany)
Sebastian Schürmann, Ruprecht-Karls-Univ. Heidelberg (Germany)
Marion Lang, German Cancer Research Ctr. (Germany)
Frederic von Wegner, Ruprecht-Karls-Univ. Heidelberg (Germany)
Oliver Friedrich, Ruprecht-Karls-Univ. Heidelberg (Germany)
Johann Engelhardt, German Cancer Research Ctr. (Germany)
Stefan W. Hell, German Cancer Research Ctr. (Germany)
Rainer H. A. Fink, Ruprecht-Karls-Univ. Heidelberg (Germany)


Published in SPIE Proceedings Vol. 6089:
Multiphoton Microscopy in the Biomedical Sciences VI
Ammasi Periasamy; Peter T. C. So, Editor(s)

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