Laboratory for Fundamental BioPhotonics, Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
Institute of Materials Science, School of Engineering, École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
Proc Natl Acad Sci U S A. 2018 Apr 17;115(16):4081-4086. doi: 10.1073/pnas.1719347115. Epub 2018 Apr 2.
Biological membranes are highly dynamic and complex lipid bilayers, responsible for the fate of living cells. To achieve this function, the hydrating environment is crucial. However, membrane imaging typically neglects water, focusing on the insertion of probes, resonant responses of lipids, or the hydrophobic core. Owing to a recent improvement of second-harmonic (SH) imaging throughput by three orders of magnitude, we show here that we can use SH microscopy to follow membrane hydration of freestanding lipid bilayers on millisecond time scales. Instead of using the UV/VIS resonant response of specific membrane-inserted fluorophores to record static SH images over time scales of >1,000 s, we SH imaged symmetric and asymmetric lipid membranes, while varying the ionic strength and pH of the adjacent solutions. We show that the nonresonant SH response of water molecules aligned by charge-dipole interactions with charged lipids can be used as a label-free probe of membrane structure and dynamics. Lipid domain diffusion is imaged label-free by means of the hydration of charged domains. The orientational ordering of water is used to construct electrostatic membrane potential maps. The average membrane potential depends quadratically on an applied external bias, which is modeled by nonlinear optical theory. Spatiotemporal fluctuations on the order of 100-mV changes in the membrane potential are seen. These changes imply that membranes are very dynamic, not only in their structure but also in their membrane potential landscape. This may have important consequences for membrane function, mechanical stability, and protein/pore distributions.
生物膜是高度动态和复杂的脂质双层，负责活细胞的命运。为了实现这一功能，水合环境至关重要。然而，膜成像通常忽略水，而是专注于探针的插入、脂质的共振响应或疏水区。由于最近二阶谐波 (SH) 成像速度提高了三个数量级，我们在这里展示了我们可以使用 SH 显微镜在毫秒时间尺度上跟踪游离脂质双层的水合作用。我们不是使用特定插入膜的荧光团的 UV/VIS 共振响应在 >1000 s 的时间尺度上记录静态 SH 图像，而是对对称和不对称脂质膜进行 SH 成像，同时改变相邻溶液的离子强度和 pH。我们表明，通过与带电脂质的电荷偶极相互作用排列的水分子的非共振 SH 响应可以用作膜结构和动力学的无标记探针。通过带电荷的域的水合作用来无标记地成像脂质域扩散。水的取向有序性用于构建静电膜电势图。平均膜电势与施加的外部偏压呈二次关系，这可以通过非线性光学理论来建模。观察到膜电势约 100 mV 的时空波动。这些变化意味着膜非常动态，不仅在其结构上，而且在其膜电势景观上也是如此。这可能对膜功能、机械稳定性和蛋白质/孔分布具有重要意义。
