4Pi-SHG imaging of mammalian myofibrillar structures

Martin Vogel; Dorothea Hahn; Sebastian Schürmann; Marion Lang; Frederic von Wegner; Oliver Friedrich; Johann Engelhardt; Stefan W. Hell; Rainer H. A. Fink

doi:10.1117/12.646638

23 February 2006 4Pi-SHG imaging of mammalian myofibrillar structures

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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Proceedings Volume 6089, Multiphoton Microscopy in the Biomedical Sciences VI; 60891Q (2006) https://doi.org/10.1117/12.646638
Event: SPIE BiOS, 2006, San Jose, California, United States

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.

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Martin Vogel, Dorothea Hahn, Sebastian Schürmann, Marion Lang, Frederic von Wegner, Oliver Friedrich, Johann Engelhardt, Stefan W. Hell, and Rainer H. A. Fink "4Pi-SHG imaging of mammalian myofibrillar structures", Proc. SPIE 6089, Multiphoton Microscopy in the Biomedical Sciences VI, 60891Q (23 February 2006); https://doi.org/10.1117/12.646638

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KEYWORDS

Second-harmonic generation

Luminescence

Microscopy

Confocal microscopy

Two photon excitation microscopy

Photonic microstructures

Single photon

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