Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein
Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein
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DOI：
10.1016/s0006-3495(02)75621-x
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发表时间：
2002-05-01
影响因子：
3.4
通讯作者：
Webb, WW
中科院分区：
文献类型：
--
作者：
Huang, SH;Heikal, AA;Webb, WW
在800 nm激发下，双光子（2P）比例氧化还原荧光法和显微镜下的吡啶核苷酸（NAD(P)H）和黄蛋白（FP）荧光已被证明是离体成年狗心肌细胞线粒体代谢状态的函数。我们测量了NAD(P)H、黄素腺嘌呤二核苷酸（FAD）和脂酰胺脱氢酶（LipDH）在720 ~ 1000 nm波长范围内的2p -激发光谱。在800 nm以下波长处，2P-激发作用截面（sigma(2P)）迅速增大，LipDH的最大sigma（2P）分别是FAD和NAD(P)H的5倍和12倍。只有FAD和LipDH能在800 nm以上的波长有效激发，在900 nm左右有较宽的2p激发带。采用2p激光扫描显微镜对分离心肌细胞的两个自身荧光光谱区（类似于410-490 nm和类似于510-650 nm）进行成像。在750 nm激发下，线粒体抑制剂NaCN和线粒体解耦剂羰基氰化物对-（三氟甲氧基）苯基腙（FCCP）诱导的荧光强度变化表明，这两个区域的荧光以NAD(P)H为主。相比之下，2P- fp荧光在900 nm激发下占优势，这与sigma（2P）测量结果一致。最后，获得了单个心脏细胞的2p -自体荧光发射光谱，结果表明所提出的2p -比率测定技术有很大的改进潜力。
Two-photon (2P) ratiometric redox fluorometry and microscopy of pyridine nucleotide (NAD(P)H) and flavoprotein (FP) fluorescence, at 800-nm excitation, has been demonstrated as a function of mitochondrial metabolic states in isolated adult dog cardiomyocytes. We have measured the 2P-excitation spectra of NAD(P)H, flavin adenine dinucleotide (FAD), and lipoamide dehydrogenase (LipDH) over the wavelength range of 720-1000 nm. The 2P-excitation action cross sections (sigma(2P)) increase rapidly at wavelengths below 800 nm, and the maximum sigma(2P) of LipDH is similar to5 and 12 times larger than those of FAD and NAD(P)H, respectively. Only FAD and LipDH can be efficiently excited at wavelengths above 800 nm with a broad 2P-excitation band around 900 nm. Two autofluorescence spectral regions (i.e., similar to410-490 nm and similar to510-650 nm) of isolated cardiomyocytes were imaged using 2P-laser scanning microscopy. At 750-nm excitation, fluorescence of both regions is dominated by NAD(P)H emission, as indicated by fluorescence intensity changes induced by mitochondrial inhibitor NaCN and mitochondria uncoupler carbonyl cyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP). In contrast, 2P-FP fluorescence dominates at 900-nm excitation, which is in agreement with the sigma(2P) measurements. Finally, 2P-autofluorescence emission spectra of single cardiac cells have been obtained, with results suggesting potential for substantial improvement of the proposed 2P-ratiometric technique.