Developmental research of a two-wavelength/simultaneous recording system of membrane potential and the coupled cellular event

使用奥林巴斯Vanox显微镜与荧光垂直照明附件，我们已经构建了一个双波长的光学记录系统，同时监测膜电位和细胞事件的变化与膜电位。该装置被设计和构造为能够进行以下程序。来自卤钨灯丝灯的光被准直，并通过显微镜上的明场冷凝器聚焦在台上的制备物上。用电位敏感染料和其他分子探针（根据需要）对制备物进行染色。通过分色镜将来自制剂的光在相应波长上分成两个组分。将两个光电探测器放置在显微镜物镜在向上（直）和矩形方向上放大图像的平面上。通常，在向上（直）方向上检测膜电位相关吸收或荧光信号，并且在矩形方向上监测膜电位耦合光散射或吸收的变化。为了选择最佳波长，在每个方向上在检测器的前面插入干涉滤光片。每个检测器的输出通过电流-电压转换器馈送到放大器，然后将放大的输出同时记录在数据记录系统上。我们希望，这里报道的方法将提高我们对各种生理功能的细胞基础的理解。

Using an Olympus Vanox Microscope with a fluorescence vertical illuminator attachment, we have constructed a two-wavelength optical recording system to simultaneously monitor changes in membrane potential and cellular events coupled with the membrane potential. This apparatus was designed and constructed to be able to make the following procedure. Light from a tungsten-halogen filament lamp was collimated, and focused on a preparation on the stage by means of the brightfield condencer on the microscope. The preparations were stained with potential-sensitive dyes and other molecular probes (as required). The light from the preparation was separated in two components by a dichroic mirror, on corresponding wavelengths. Two photodetectors were placed at the planes where the microscope objective makes magnified images in the upward (straight) and rectangular directions. Usually, the membrane potential-related absorption or fluorescence signals were detected in the upward (straight) direction and the changes in the membrane potential-coupled light scattering or absorption were monitored in the rectangular direction. In order to select an optimal wavelength, an interference filter was inserted in the front of the detector in each direction. The output of each detector was fed to an amplifier via a current-to-voltage converter, and then the amplified outputs were recorded simultaneously on a data recording system. We hope that the method reported here would enhance our understanding of the cellular basis of various physiological functions.