Optical single-channel recording by imaging Ca2+ flux through individual ion channels:: theoretical considerations and limits to resolution
Optical single-channel recording by imaging Ca2+ flux through individual ion channels:: theoretical considerations and limits to resolution
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DOI：
10.1016/j.ceca.2004.10.008
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发表时间：
2005-04-01
期刊：
影响因子：
4
通讯作者：
Parker, I
中科院分区：
文献类型：
--
作者：
Shuai, JW;Parker, I
显微镜和荧光指示剂的最新发展使得通过成像单个离子通道的钙离子通量来监测膜离子通道的活性和定位成为可能。这种光学方法与电生理单通道技术相比具有优势，因为它们侵入性较小，提供空间信息，并且可以同时和独立地监控数百个通道。然而，它们的动力学分辨率还不能达到膜片钳记录的水平。为了帮助理解决定单通道Ca~(2+)荧光信号(SCCaFT)时间分辨率和噪声水平的过程，我们模拟了开放通道口周围存在的Ca~(2+)离子的微域和与Ca~(2+)结合的指示剂染料。此外，为了帮助改进的光学技术的发展，我们模拟了SCCaFT的幅度和动力学随成像体积、指示剂浓度、亲和力和迁移率以及内源和外源钙缓冲液的存在等参数的变化。结果表明，在最佳条件下，包括使用共焦或全内反射显微镜从亚毫微升体积成像，SCCaFTs应能在信噪比为10的情况下解析短至1ms的通道开口。
Recent developments in microscopy and fluorescent indicators now make it possible to monitor the activity and localization of membrane ion channels by imaging Ca2+ flux through individual channels. Such optical approaches have advantages over electrophysiological single-channel techniques in that they are less invasive, provide spatial information and can simultaneously and independently monitor hundreds of channels. However, their kinetic resolution does not yet approach that of patch-clamp recordings. To help understand the processes that determine the temporal resolution and noise level of single-channel Ca2+ fluorescence signals (SCCaFTs), we simulated the microdomains of Ca2+ ions and Ca2+-bound indicator dye that exist around the mouth of an open channel. Further, as an aid to development of improved optical techniques, we modeled the dependence of the amplitude and kinetics of SCCaFTs on parameters such as the imaging volume, the indicator concentration, affinity and mobility, and the presence of endogenous and exogenous Ca2+ buffers. The results indicate that under optimal conditions, including the use of confocal or total-internal reflection microscopy to image from sub-femtolitre volumes, SCCaFTs should resolve channel openings as brief as 1 ms with a signal-to-noise ratio >10. (C) 2005 Elsevier Ltd. All rights reserved.