Millard A C, Jin L, Wuskell J P, Boudreau D M, Lewis A, Loew L M
Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, 06030, USA.
J Membr Biol. 2005 Nov;208(2):103-11. doi: 10.1007/s00232-005-0823-y.
Second harmonic generation (SHG) imaging microscopy is an important emerging technique for biological research, complementing existing one- and two-photon fluorescence (2PF) methods. A non-linear phenomenon employing light from mode-locked Ti:sapphire or fiber-based lasers, SHG results in intrinsic optical sectioning without the need for a confocal aperture. Furthermore, as a second-order process SHG is confined to loci lacking a center of symmetry, a constraint that is readily satisfied by lipid membranes with only one leaflet stained by a dye. Of particular interest is "resonance-enhanced" SHG from styryl dyes in cellular membranes and the possibility that SHG is sensitive to transmembrane potential. We have previously confirmed this, using simultaneous voltage-clamping and non-linear imaging of cells to find that SHG is up to four times more sensitive to potential than fluorescence. In this work, we have extended these results in two directions. First, with a range of wavelengths available from a mode-locked Ti:sapphire laser and a fiber-based laser, we have more fully investigated SHG and 2PF voltage-sensitivity from ANEP and ASTAP chromophores, obtaining SHG sensitivity spectra that are consistent with resonance enhancements. Second, we have modified our system to coordinate the application of voltage-clamp steps with non-linear image acquisition to more precisely characterize the time dependence of SHG and 2PF voltage sensitivity, finding that, at least for some dyes, SHG responds more slowly than fluorescence to changes in transmembrane potential.
二次谐波产生（SHG）成像显微镜是生物研究中一项重要的新兴技术，它是对现有的单光子和双光子荧光（2PF）方法的补充。SHG是一种利用锁模钛宝石激光器或光纤激光器发出的光的非线性现象，它无需共焦孔径即可实现固有光学切片。此外，作为一种二阶过程，SHG局限于缺乏对称中心的位点，对于仅用一种染料染色一个小叶的脂质膜来说，这一限制很容易满足。特别令人感兴趣的是细胞膜中苯乙烯基染料产生的“共振增强”SHG以及SHG对跨膜电位敏感的可能性。我们之前已经证实了这一点，通过同时对细胞进行电压钳制和非线性成像发现，SHG对电位的敏感度比荧光高四倍。在这项工作中，我们从两个方向扩展了这些结果。第一，利用锁模钛宝石激光器和光纤激光器提供的一系列波长，我们更全面地研究了ANEP和ASTAP发色团的SHG和2PF电压敏感度，获得了与共振增强一致的SHG敏感度光谱。第二，我们改进了我们的系统，以协调电压钳制步骤的应用与非线性图像采集，从而更精确地表征SHG和2PF电压敏感度的时间依赖性，发现至少对于某些染料，SHG对跨膜电位变化的响应比荧光慢。
