Optical imaging and functional characterization of the transverse tubular system of mammalian muscle fibers using the potentiometric indicator di-8-ANEPPS
Optical imaging and functional characterization of the transverse tubular system of mammalian muscle fibers using the potentiometric indicator di-8-ANEPPS
复制标题
DOI：
10.1007/s00232-005-0825-9
复制
发表时间：
2005-11-01
影响因子：
2.4
通讯作者：
Vergara, JL
中科院分区：
文献类型：
--
作者：
DiFranco, M;Capote, J;Vergara, JL
电位法染料是研究直接电记录无法接触到的隔室的膜电位变化的有用工具。过去，我们结合电生理和光学技术，通过吸光度和荧光电位染料进行研究。两栖骨骼肌纤维中横管系统的电学特性。本文利用荧光电位指示剂di-8-ANEPPS对哺乳动物骨骼肌纤维横管系统的结构和功能特性进行了研究。活的肌肉纤维的双光子激光扫描共聚焦荧光图像表明，在伸展到肌节长度达3.5微米的肌肉纤维中，Z线两侧连续的横管之间的距离保持相对恒定。此外，将双微电极电生理技术与显微荧光光谱和成像相结合，使我们能够比较静止纤维中di-8-ANEPPS荧光的光谱特性，以及在刺激纤维中记录的荧光瞬变的光谱特性。我们发现，尽管指示剂的激发峰和发射峰分别位于470 nm和588 nm，但当使用滤光片选择530-550mN波段的激发波长和590 nm以上的发射波长时，荧光瞬变显示出最佳的分数变化(13%/100 mV)。在这种情况下，强直刺激纤维和电压钳实验的结果有力地表明，尽管哺乳动物纤维中di-8-ANEPPS的瞬变动力学非常快，与表膜电记录相似，但它们来自横管系统膜。
Potentiometric dyes are Useful tools for studying membrane potential changes from compartments inaccessible to direct electrical recordings. In the past, we have combined electrophysiological and optical techniques to investigate, by using absorbance and fluorescence potentiometric dyes.. the electrical properties of the transverse tubular system in amphibian skeletal muscle fibers. In this paper we expand on recent observations using the fluorescent potentiometric indicator di-8-ANEPPS to investigate structural and functional properties of the transverse tubular system in mammalian skeletal muscle fibers. Two-photon laser scanning confocal fluorescence images of live Muscle fibers suggest that the distance between consecutive rows of transverse tubules flanking the Z-lines remains relatively constant in Muscle fibers stretched to attain sarcomere lengths of up to 3.5 mu m. Furthermore, the combined use of two-microelectrode electrophysiological techniques with microscopic fluorescence spectroscopy and imaging allowed us to compare the spectral properties of di-8-ANEPPS fluorescence in fibers at rest, with those of fluorescence transients recorded in stimulated fibers. We found that although the indicator has excitation and emission peaks at 470 and 588 nm, respectively, fluorescence transients display optimal fractional changes (13%/100 mV) when using filters to select excitation wavelengths in the 530-550 mn band and emissions beyond 590 nm. Under these conditions, results from tetanically stimulated fibers and from voltage-clamp experiments Suggest strongly that, although the kinetics of di-8-ANEPPS transients in mammalian fibers are very rapid and approximate those of the surface membrane electrical recordings, they arise from the transverse tubular system membranes.