Intracellular fluorescent probe concentrations by confocal microscopy
Intracellular fluorescent probe concentrations by confocal microscopy
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DOI：
10.1016/s0006-3495(98)77607-6
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发表时间：
1998-10-01
影响因子：
3.4
通讯作者：
Loew, LM
中科院分区：
文献类型：
--
作者：
Fink, C;Morgan, F;Loew, LM
描述了一种利用共聚焦显微镜的光学切片特性来测量细胞内荧光分子的绝对浓度和相对浓度的通用方法。对于实质上大于点扩散函数的细胞内隔间，荧光强度与荧光团的浓度成正比。对于小隔室，荧光强度被来自隔室外区域的贡献所稀释。这种稀释的校正可以通过基于在通过计算合成的细胞或细胞器的计算合成模型中找到的强度分布的校准来估计，该细胞或细胞器已经通过与显微镜的点扩散函数卷积而变得模糊。该方法用四个测试案例来说明：估计荧光钙指示剂的细胞内浓度；估计内质网INSP(3)受体的神经细胞突起和胞体之间的相对分布；估计缓激肽受体沿神经元细胞表面的分布；以及作为分析线粒体膜电位的手段，在线粒体和胞浆之间的电位染料的相对分布。
A general method is described that; takes advantage of the optical sectioning properties of a confocal microscope to enable measurement of both absolute and relative concentrations of fluorescent molecules inside cells. For compartments within cells that are substantially larger than the point spread function, the fluorescence intensity is simply proportional to the concentration of the fluorophore. For small compartments, the fluorescence intensity is diluted by contributions from regions outside the compartment. Corrections for this dilution can be estimated via calibrations that are based on the intensity distribution found in a computationally synthesized model for a cell or organelle that has been blurred by convolution with the microscope point spread function. The method is illustrated with four test cases: estimation of intracellular concentration of a fluorescent calcium indicator; estimation of the relative distribution between the neurite and soma of a neuronal cell of the InsP(3) receptor on the endoplasmic reticulum; estimation of the distribution of the bradykinin receptor along the surface of a neuronal cell; and relative distribution of a potentiometric dye between the mitochondria and cytosol as a means of assaying mitochondrial membrane potential.