Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues
Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues
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DOI：
10.1016/s0006-3495(02)75414-3
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发表时间：
2002-01-01
影响因子：
3.4
通讯作者：
Mohler, WA
中科院分区：
文献类型：
--
作者：
Campagnola, PJ;Millard, AC;Mohler, WA
我们发现，几个关键的内源性蛋白质结构引起强烈的二次谐波产生（SHG）-非吸收倍频的激发激光线。因此，激光扫描系统上的二次谐波成像显微镜（SHIM）被证明是一种强大的，独特的工具，用于活细胞和组织结构的高分辨率，高对比度，三维研究。与荧光不同，SHG没有固有的光漂白或毒性，并且不需要外源标记。与偏光显微镜不同，SHIM提供了内在的共焦性和复杂组织的深层切片。在这项研究中，我们证明了清晰的SHIM光学切片内未固定，未染色厚标本。将SHIM和双光子激发荧光（TPEF）结合在一个双模式非线性显微镜中，以阐明活细胞和组织中SHG的分子来源。SHG不仅来源于结缔组织和肌肉粗丝内的卷曲螺旋复合物，而且来源于间期细胞和有丝分裂细胞内的微管阵列。这两个偏振依赖性和局部对称性，消除效应的SHG允许的信号从物种产生的二次谐波被解码，通过比率相关与TPEF，产生信息的光学分辨率以下的局部结构。这些组织内的SHG的物理起源被解决，并归因于激光与偶极蛋白质结构的相互作用，增强了蛋白质螺旋的固有手性。
We find that several key endogenous protein structures give rise to intense second-harmonic generation (SHG)-nonabsorptive frequency doubling of an excitation laser line. Second-harmonic imaging microscopy (SHIM) on a laser-scanning system proves, therefore, to be a powerful,and unique tool for high-resolution, high-contrast, three-dimensional studies of live cell and tissue architecture. Unlike fluorescence, SHG suffers no inherent photobleaching or toxicity and does not require exogenous labels. Unlike polarization microscopy, SHIM provides intrinsic confocality and deep sectioning in complex tissues. In this study, we demonstrate the clarity of SHIM optical sectioning within unfixed, unstained thick specimens. SHIM and two-photon excited fluorescence (TPEF) were combined in a dual-mode nonlinear microscopy to elucidate the molecular sources of SHG in live cells and tissues. SHG arose not only from coiled-coil complexes within connective tissues and muscle thick filaments, but also from microtubule arrays within interphase and mitotic cells. Both polarization dependence and a local symmetry, cancellation effect of SHG allowed the signal from species generating the second harmonic to be decoded, by ratiometric correlation with TPEF, to yield information on local structure below optical resolution. The physical origin of SHG within these tissues is addressed and is attributed to the laser interaction with dipolar protein structures that is enhanced by the intrinsic chirality of the protein helices.