Sinha S R, Saggau P
Division of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.
Methods. 1999 Jun;18(2):204-14, 175. doi: 10.1006/meth.1999.0773.
Optical recording techniques provide a constantly evolving and increasingly powerful set of tools for investigations of cellular physiology. These techniques rely on the use of optical indicators, molecules that change their optical properties depending on the cellular parameter of interest. In this paper we discuss some of the general considerations involved in recording optical signals from multiple indicators. Specifically, we describe a technique for simultaneously recording transients of membrane potential and intracellular calcium concentration, two parameters that have a very complex interrelationship in neuronal functioning. This technique relies on the use of two fluorescent indicators (the voltage-sensitive dye RH-414 and the calcium-sensitive dye Calcium Orange) that have overlapping excitation spectra but separable emission spectra. This fact, in combination with the use of fast, spatially resolving photodetectors (10 x 10-element photodiode matrices), allows for truly simultaneous recording of these transients from brain slices with high spatial ( approximately 200 x 200 microm with a 10x microscope objective) and temporal ( approximately 500 micros) resolution. Furthermore, the quality of the signals obtained is sufficient to allow for recording of spontaneous synchronized activity such as epileptiform activity induced by the potassium channel blocker 4-aminopyridine. The nature of the signals obtained by these indicators recorded from guinea pig hippocampal slices and some applications of this technique are discussed.
光学记录技术为细胞生理学研究提供了一套不断发展且日益强大的工具。这些技术依赖于光学指示剂的使用，光学指示剂是一类分子，其光学特性会根据感兴趣的细胞参数而改变。在本文中，我们讨论了从多个指示剂记录光学信号时涉及的一些一般注意事项。具体而言，我们描述了一种同时记录膜电位瞬变和细胞内钙浓度瞬变的技术，这两个参数在神经元功能中存在非常复杂的相互关系。该技术依赖于使用两种荧光指示剂（电压敏感染料RH - 414和钙敏感染料钙黄绿素），它们具有重叠的激发光谱但发射光谱可分离。这一事实，结合使用快速、具有空间分辨能力的光电探测器（10×10元素光电二极管矩阵），能够以高空间分辨率（使用10倍显微镜物镜时约为200×200微米）和时间分辨率（约500微秒）从脑片真正同时记录这些瞬变。此外，获得的信号质量足以记录自发同步活动，如钾通道阻滞剂4 - 氨基吡啶诱导的癫痫样活动。本文还讨论了从豚鼠海马脑片记录这些指示剂获得的信号性质以及该技术的一些应用。
