Using total fluorescence increase (signal mass) to determine the Ca2+ current underlying localized Ca2+ events.
Using total fluorescence increase (signal mass) to determine the Ca2+ current underlying localized Ca2+ events.
复制标题
DOI：
10.1085/jgp.200409066
复制
发表时间：
2004-09
期刊：
影响因子：
--
通讯作者：
Singer JJ
中科院分区：
文献类型：
--
作者：
Zou H;Lifshitz LM;Tuft RA;Fogarty KE;Singer JJ
通过成像（使用 Fluo-3）单通道 Ca2+ 荧光瞬变 (SCCaFT) 探索仅使用宽场荧光图像确定局部 Ca2+ 流入的可行性，因为 Ca2+ 通过 Ca2+ 渗透性离子通道的单个开口进入，同时记录单一通道电流。由于使用宽视场光学器件获得的图像是焦内和离焦光的综合，因此可以通过将所有像素中 Ca2+ 流入导致的荧光增加相加，直接从图像中测量与 SCCaFT 相关的总荧光增加（ΔFtotal 或“信号质量”）。不需要从共焦线扫描图像获得信号质量所需的假设。使用二维和三维成像表明 ΔFtotal 基本上与通道相对于显微镜焦平面的位置无关。当 Ca2+ 是内向电流的唯一电荷载体时，使用质膜咖啡因激活的阳离子通道的 SCCaFT 获得 Ca2+ 流入量与 ΔFtotal 之间的关系。人们发现这种关系是线性的，斜率（或转换因子）的值受到特定成像系统设置、实验条件和荧光指示剂特性（包括其与其他细胞缓冲液的结合能力）的影响。转换因子用于估计通过近生理盐水中咖啡因激活通道的 Ca2+ 电流，并估计内源缓冲液结合能力。此外，它还可以更准确地估计同一制剂中通过兰尼碱受体从细胞内储存的 Ca2+ 释放产生的 Ca2+ 火花所引起的 Ca2+ 电流。
The feasibility of determining localized Ca2+ influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca2+ fluorescence transients (SCCaFTs), due to Ca2+ entry through single openings of Ca2+-permeable ion channels, while recording unitary channel currents. Since the image obtained with wide-field optics is an integration of both in-focus and out-of-focus light, the total fluorescence increase (ΔFtotal or “signal mass”) associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca2+ influx in all of the pixels. The assumptions necessary for obtaining the signal mass from confocal linescan images are not required. Two- and three-dimensional imaging was used to show that ΔFtotal is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca2+ influx and ΔFtotal was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca2+ was the only charge carrier of the inward current. This relationship was found to be linear, with the value of the slope (or converting factor) affected by the particular imaging system set-up, the experimental conditions, and the properties of the fluorescent indicator, including its binding capacity with respect to other cellular buffers. The converting factor was used to estimate the Ca2+ current passing through caffeine-activated channels in near physiological saline and to estimate the endogenous buffer binding capacity. In addition, it allowed a more accurate estimate of the Ca2+ current underlying Ca2+ sparks resulting from Ca2+ release from intracellular stores via ryanodine receptors in the same preparation.
登录
查看更多内容
影响因子：
56.9
作者：
NELSON, MT;CHENG, H;LEDERER, WJ
通讯作者：
LEDERER, WJ
DOI：
10.1085/jgp.113.3.373
发表时间：
1999-03
期刊：
The Journal of general physiology
影响因子：
--
作者：
Cannell MB;Soeller C
通讯作者：
Soeller C
DOI：
10.1085/jgp.116.6.735
发表时间：
2000-12
期刊：
The Journal of general physiology
影响因子：
--
作者：
Ohyama T;Hackos DH;Frings S;Hagen V;Kaupp UB;Korenbrot JI
通讯作者：
Korenbrot JI
影响因子：
5.5
作者：
Bootman, M;Niggli, E;Lipp, P
通讯作者：
Lipp, P
影响因子：
4
作者：
Daub, B;Ganitkevich, VY
通讯作者：
Ganitkevich, VY