Jonas E A, Knox R J, Kaczmarek L K
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Neuron. 1997 Jul;19(1):7-13. doi: 10.1016/s0896-6273(00)80343-8.
A method is outlined for obtaining giga-ohm seals on intracellular membranes in intact cells. The technique employs a variant of the patch-clamp technique: a concentric electrode arrangement protects an inner patch pipette during penetration of the plasma membrane, after which a seal can be formed on an internal organelle membrane. Using this technique, successful recordings can be obtained with the same frequency as with conventional patch clamping. To localize the position of the pipette within cells, lipophilic fluorescent dyes are included in the pipette solution. These dyes stain the membrane of internal organelles during seal formation and can then be visualized by video-enhanced or confocal imaging. The method can detect channels activated by inositol trisphosphate, as well as other types of intracellular membrane ion channel activity, and should facilitate studies of internal membranes in intact neurons and other cell types.
本文概述了一种在完整细胞的细胞内膜上获得千兆欧密封的方法。该技术采用了膜片钳技术的一种变体：同心电极装置在质膜穿透过程中保护内部膜片吸管，之后可在内部细胞器膜上形成密封。使用该技术，可以获得与传统膜片钳记录相同频率的成功记录。为了在细胞内定位吸管的位置，亲脂性荧光染料被包含在吸管溶液中。这些染料在密封形成过程中对内部细胞器的膜进行染色，然后可以通过视频增强或共聚焦成像进行观察。该方法可以检测由肌醇三磷酸激活的通道以及其他类型的细胞内膜离子通道活性，并且应该有助于对完整神经元和其他细胞类型的内膜进行研究。
