Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: Topology and optical inner filters
Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: Topology and optical inner filters
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DOI：
10.1529/biophysj.104.053165
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发表时间：
2005-03-01
影响因子：
3.4
通讯作者：
Balaban, RS
中科院分区：
文献类型：
--
作者：
Rothstein, EC;Carroll, S;Balaban, RS
双光子激发荧光显微镜（TPEFM）允许在活的动物细胞间事件的拓扑结构的调查。采用TPEFM监测小鼠骨骼肌线粒体还原性烟酰胺腺嘌呤二核苷酸（NAD(P) H）在体内的分布。在710 - 720 nm激发下监测NAD(P) H荧光发射（类似于460 nm）。高分辨率TPEFM图像从胫骨前肌表面采集至150mm深度。NAD(P) H荧光图像显示亚细胞结构与肌层下、血管周围、肌间和核旁线粒体一致。利用NAD(P) H荧光信号中线粒体的分布和含量，可以在体内分型IIB和IIA/D纤维。在IIB纤维类型中，肌节间线粒体集中在z线上，形成间隔为1个肌节的周期性模式（2.34 +/- 0.17 μ m）。初级内滤效应几乎等同于水，但次级内滤效应非常显著，并动态影响NAD(P) H荧光信号的发射频率和振幅。这些数据证明了在骨骼肌中使用NAD(P) H TPEFM来表征活体小鼠线粒体拓扑结构和代谢功能的可行性，并突出了其复杂性。
Two-photon excitation fluorescence microscopy (TPEFM) permits the investigation of the topology of intercellular events within living animals. TPEFM was used to monitor the distribution of mitochondrial reduced nicotinamide adenine dinucleotide ( NAD(P) H) in murine skeletal muscle in vivo. NAD( P) H fluorescence emission was monitored (similar to460 nm) using 710 - 720 nm excitation. High-resolution TPEFM images were collected up to a depth of 150 mm from the surface of the tibialis anterior muscle. The NAD( P) H fluorescence images revealed subcellular structures consistent with subsarcolemmal, perivascular, intersarcomeric, and paranuclear mitochondria. In vivo fiber typing between IIB and IIA/D fibers was possible using the distribution and content of mitochondria from the NAD( P) H fluorescence signal. The intersarcomeric mitochondria concentrated at the Z-line in the IIB fiber types resulting in a periodic pattern with a spacing of one sarcomere (2.34 +/- 0.17 mum). The primary inner filter effects were nearly equivalent to water, however, the secondary inner filter effects were highly significant and dynamically affected the observed emission frequency and amplitude of the NAD( P) H fluorescence signal. These data demonstrate the feasibility, and highlight the complexity, of using NAD( P) H TPEFM in skeletal muscle to characterize the topology and metabolic function of mitochondria within the living mouse.