4Pi-SHG imaging of mammalian myofibrillar structures

Vogel M, Hahn D, Schürmann S, Lang M, von Wegner F, Friedrich O, Engelhardt J, Hell S, Fink R (2006)

Publication Status: Published

Publication Type: Conference contribution, Conference Contribution

Publication year: 2006

Book Volume: 6089

Article Number: 60891Q

Event location: San Jose, CA

ISBN: 9780819461315

DOI: 10.1117/12.646638

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 mu;m in length, (ii) consist of single myofibrils of small total diameter of approximately 1 mu;m 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 fractionation (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.

Authors with CRIS profile

Sebastian Schürmann Lehrstuhl für Medizinische Biotechnologie Oliver Friedrich Lehrstuhl für Medizinische Biotechnologie

Involved external institutions

Deutsches Krebsforschungszentrum (DKFZ) DE Germany (DE) Ruprecht-Karls-Universität Heidelberg DE Germany (DE) Goethe-Universität Frankfurt am Main DE Germany (DE) Max-Planck-Institut für biophysikalische Chemie (Karl-Friedrich-Bonhoeffer-Institut) / Max Planck Institute for Biophysical Chemistry DE Germany (DE)

How to cite

APA:

Vogel, M., Hahn, D., Schürmann, S., Lang, M., von Wegner, F., Friedrich, O.,... Fink, R. (2006). 4Pi-SHG imaging of mammalian myofibrillar structures. In Proceedings of the Multiphoton Microscopy in the Biomedical Sciences VI. San Jose, CA.

MLA:

Vogel, Martin, et al. "4Pi-SHG imaging of mammalian myofibrillar structures." Proceedings of the Multiphoton Microscopy in the Biomedical Sciences VI, San Jose, CA 2006.

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