Quantitative two-photon Ca2+ imaging via fluorescence lifetime analysis
Quantitative two-photon Ca2+ imaging via fluorescence lifetime analysis
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DOI：
10.1016/j.ceca.2006.03.006
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发表时间：
2006-07-01
期刊：
影响因子：
4
通讯作者：
Eilers, J
中科院分区：
文献类型：
--
作者：
Wilms, CD;Schmidt, H;Eilers, J
双光子显微镜（TPM）通过允许完整组织和活生物体的深度记录而彻底改变了Ca 2+成像。然而，TPM的一个严重限制是缺乏一种准确和直接的方法来定量Ca 2+信号，这种能力成为荧光显微镜中的一个宝贵工具。在这里，我们提出了时间相关的荧光寿命成像（tcFLIM）作为一个比率的方法在TPM中的Ca 2+信号的定量。Ca 2+指示剂染料俄勒冈州绿色BAPTA-1（OGB-1）的荧光寿命可以使用类似于TPM中使用的80 MHz激发脉冲来记录。它显示了Ca 2+的依赖性，可以解释的Ca 2+的亲和力，光谱特性和纯度的染料。逐像素的寿命记录，由激光扫描显微镜控制，允许定量Ca 2+成像在全帧和行扫描模式。虽然我们专注于高亲和力的Ca 2+指示剂OGB-1。我们的基于tcFLIM的定量通常适用于其它Ca 2+染料和荧光指示剂。(c)2006爱思唯尔有限公司保留所有权利。
Two-photon microscopy (TPM) revolutionized Ca2+ imaging by allowing recordings in the depth of intact tissue and live organisms. A serious limitation in TPM, however, is the lack of an accurate and straightforward approach for the quantification of Ca2+ signals, an ability that became an invaluable tool in fluorescence microscopy. Here, we present time-correlated fluorescence lifetime imaging (tcFLIM) as a ratiometric method for the quantification of Ca2+ signals in TPM. The fluorescence lifetime of the Ca2+ indicator dye Oregon Green BAPTA-1 (OGB-1) can be recorded using the similar to 80 MHz excitation pulses utilized in TPM. It shows a Ca2+ dependence that can be explained by the Ca2+-affinity, spectral properties and purity of the dye. Pixel-wise lifetime recordings, controlled by a laser-scanning microscope, allowed quantitative Ca2+ imaging in full-frame and linescan mode. Although we focused on the high-affinity Ca2+ indicator OGB-1. our tcFLIM-based quantification is applicable to other Ca2+ dyes and to fluorescence indicators in general. (c) 2006 Elsevier Ltd. All rights reserved.