The Epidermal Ca2+ Gradient: Measurement Using the Phasor Representation of Fluorescent Lifetime Imaging
The Epidermal Ca2+ Gradient: Measurement Using the Phasor Representation of Fluorescent Lifetime Imaging
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DOI：
10.1016/j.bpj.2009.10.055
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发表时间：
2010-03-03
影响因子：
3.4
通讯作者：
Mauro, T.
中科院分区：
文献类型：
--
作者：
Celli, A.;Sanchez, S.;Mauro, T.
在各种组织和器官中都发现了离子梯度。在本报告中，我们将荧光寿命成像数据的相量表示应用于组织中离子浓度的定量研究，克服了组织厚度、离子敏感染料的浓度伪影和跨非均匀组织的校准等技术问题。我们使用表皮作为模型系统，因为该器官中的Ca2+梯度先前已被证明可以控制表皮通透性屏障的分化和形成的基本生物过程。这里描述的方法允许更好的Ca2+存储的定位比以前的研究中使用的，并揭示了在表皮中测量的游离Ca2+的大部分来自细胞内的Ca2+存储，如高尔基体和内质网，细胞外Ca2+对表皮Ca2+梯度的贡献相对较小。由于双光子显微镜的高空间分辨率，我们能够测量基底角化细胞中细胞间平均钙浓度的显著异质性。这一发现，在以前的研究中没有报道，质疑长期持有的假设，即角化细胞增加细胞内Ca2+停止增殖，并被动地分化以响应细胞外Ca2+的变化。使用这种方法获得的实验结果说明了本报告中概述的实验和分析技术的力量。我们的方法可以用于机制研究，以解决表皮Ca2+梯度的形成、维持和功能，并且它应该广泛适用于研究其他具有离子梯度的组织。
Ionic gradients are found across a variety of tissues and organs. In this report, we apply the phasor representation of fluorescence lifetime imaging data to the quantitative study of ionic concentrations in tissues, overcoming technical problems of tissue thickness, concentration artifacts of ion-sensitive dyes, and calibration across inhomogeneous tissue. We used epidermis as a model system, as Ca2+ gradients in this organ have been shown previously to control essential biologic processes of differentiation and formation of the epidermal permeability barrier. The approach described here allowed much better localization of Ca2+ stores than those used in previous studies, and revealed that the bulk of free Ca2+ measured in the epidermis comes from intracellular Ca2+ Stores such as the Golgi and the endoplasmic reticulum, with extracellular Ca2+ making a relatively small contribution to the epidermal Ca2+ gradient. Due to the high spatial resolution of two-photon microscopy, we were able to measure a marked heterogeneity in average calcium concentrations from cell to cell in the basal keratinocytes. This finding, not reported in previous studies, calls into question the long-held hypothesis that keratinocytes increase intracellular Ca2+ cease proliferation, and differentiate passively in response to changes in extracellular Ca2+. The experimental results obtained using this approach illustrate the power of the experimental and analytical techniques outlined in this report. Our approach can be used in mechanistic studies to address the formation, maintenance, and function of the epidermal Ca2+ gradient, and it should be broadly applicable to the study of other tissues with ionic gradients.