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.
In confocal microscopy manifold combinations of dyes are used. The entire wavelength range from near-ultraviolet to near-infrared is used to excite these dyes. Their emission is then detected primarily in the visible range. In addition to the number of dyes, the number of possible excitation laser lines is already large and constantly increasing. Due to the almost unlimited number of possible combinations, programmable devices are required. This is true for excitation-modules, beam-splitters and detection modules. Systems using filters with fixed spectral properties can practically fulfill the requirements for only a very small subset of applications. Programmable devices have been realized for excitation using Acousto Optical Tunable Filters (AOTF's). Freely definable spectral detectors are available for a perfect adaptation to the desired emission bands. The missing link for a completely filter-free design is now introduced using an Acousto Optical Beam Splitter (AOBS).
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