Institute of Neurobiology, Faculty of Mathematics and Natural Sciences, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.
Institute of Complex Systems 4 (ICS-4), Zelluläre Biophysik, Forschungszentrum Jülich, Jülich, Germany.
J Gen Physiol. 2019 Nov 4;151(11):1319-1331. doi: 10.1085/jgp.201912404. Epub 2019 Oct 9.
Fluorescence lifetime imaging microscopy (FLIM) with fluorescent ion sensors enables the measurement of ion concentrations based on the detection of photon emission events after brief excitation with a pulsed laser source. In contrast to intensity-based imaging, it is independent of dye concentration, photobleaching, or focus drift and has thus been successfully employed for quantitative analysis of, e.g., calcium levels in different cell types and cellular microdomains. Here, we tested the suitability of CoroNaGreen for FLIM-based determination of sodium concentration ([Na]) inside cells. In vitro measurements confirmed that fluorescence lifetimes of CoroNaGreen (CoroNaFL) increased with increasing [Na]. Moreover, CoroNaFL was largely independent of changes in potassium concentration or viscosity. Changes in pH slightly affected FL in the acidic range (pH ≤ 5.5). For intracellular determination of [Na], HEK293T cells were loaded with the membrane-permeable form of CoroNaGreen. Fluorescence decay curves of CoroNaGreen, derived from time-correlated single-photon counting, were approximated by a bi-exponential decay. In situ calibrations revealed a sigmoidal dependence of CoroNaFL on [Na] between 0 and 150 mM, exhibiting an apparent of ∼80 mM. Based on these calibrations, a [Na] of 17.6 mM was determined in the cytosol. Cellular nuclei showed a significantly lower [Na] of 13.0 mM, whereas [Na] in perinuclear regions was significantly higher (26.5 mM). Metabolic inhibition or blocking the Na/K-ATPase by removal of extracellular K caused significant [Na] increases in all cellular subcompartments. Using an alternative approach for data analysis ("Ratio FLIM") increased the temporal resolution and revealed a sequential response to K removal, with cytosolic [Na] increasing first, followed by the nucleus and finally the perinuclear regions. Taken together, our results show that CoroNaGreen is suitable for dynamic, FLIM-based determination of intracellular [Na]. This approach thus represents a valuable tool for quantitative determination of [Na] and changes thereof in different subcellular compartments.
荧光寿命成像显微镜（FLIM）结合荧光离子传感器，可以通过短暂激发脉冲激光源后检测光子发射事件来测量离子浓度。与基于强度的成像不同，它不受染料浓度、光漂白或焦点漂移的影响，因此已成功用于定量分析不同细胞类型和细胞微区的钙水平等。在这里，我们测试了 CoroNaGreen 用于基于 FLIM 的细胞内钠离子浓度 ([Na]) 测定的适用性。体外测量证实，CoroNaGreen 的荧光寿命（CoroNaFL）随 [Na] 的增加而增加。此外，CoroNaFL 基本不受钾浓度或粘度变化的影响。pH 值的变化在酸性范围内（pH ≤ 5.5）对 FL 稍有影响。为了在细胞内测定 [Na]，将 HEK293T 细胞加载膜通透性形式的 CoroNaGreen。通过时间相关单光子计数得到的 CoroNaGreen 荧光衰减曲线用双指数衰减来近似。原位校准表明，CoroNaFL 与 0 至 150 mM 之间的 [Na] 呈 S 型依赖关系，表观 约为 80 mM。基于这些校准，在细胞质中确定了 [Na] 为 17.6 mM。细胞核显示出明显较低的 [Na]（13.0 mM），而核周区的 [Na] 明显较高（26.5 mM）。代谢抑制或通过去除细胞外 K 阻断 Na/K-ATP 酶会导致所有细胞亚区的 [Na] 显著增加。使用数据分析的替代方法（“Ratio FLIM”）提高了时间分辨率，并揭示了对 K 去除的顺序响应，细胞质 [Na] 首先增加，随后是细胞核，最后是核周区。总之，我们的结果表明 CoroNaGreen 适合用于动态、基于 FLIM 的细胞内 [Na] 测定。这种方法因此代表了定量测定不同亚细胞区室中 [Na] 和变化的有价值的工具。
