Calibration and validation of an optical sensor for intracellular oxygen measurements.
Calibration and validation of an optical sensor for intracellular oxygen measurements.
复制标题
用于细胞内氧测量的光学传感器的校准和验证。
DOI：
10.1117/1.3116714
复制
发表时间：
2009
影响因子：
3.5
通讯作者：
Mycek,Mary-Ann
中科院分区：
文献类型：
--
作者：
Sud,Dhruv;Mycek,Mary-Ann
荧光光学传感器在体内进行精确、定量的细胞内测量的校准，缺乏适当模拟细胞溶胶分子复杂性的代表性介质。我们提出了一种通过荧光寿命成像显微镜（FLIM）精确检测细胞内氧的新方案，使用细胞裂解液-FLIM测量来纠正荧光氧传感器的玻璃校准，我们描述了电子顺磁共振（EPR）验证研究。Lysate-FLIM研究提供了生化信息，而EPR提供了细胞内氧估计的“金标准”。在活的人类正常鳞状和腺癌食管上皮细胞中评估氧水平，并观察到光学方案和EPR得出的氧水平之间的良好一致性。提出的方案引入了活细胞系的概念，作为估计其他细胞系中未知氧水平的参考，并考虑到不同细胞系之间的高度可变性。
Calibration of fluorescent optical sensors for accurate, quantitative intracellular measurementsin vivosuffers from lack of a representative medium that appropriately simulates the molecular complexity of the cytosol. We present a novel protocol for accurate intracellular oxygen sensing via fluorescence lifetime imaging microscopy (FLIM) using cell lysate-FLIM measurements to correct thein vitrocalibration of a fluorescent oxygen sensor, and we describe electron paramagnetic resonance (EPR) validation studies. Lysate-FLIM studies provided biochemical information, while EPR provided a “gold standard” for intracellular oxygen estimation. Oxygen levels were evaluated in living human normal squamous and adenocarcinoma esophageal epithelial cells, and good agreement was observed between oxygen levels derived from the optical protocol and EPR. The proposed protocol introduces the concept of a living cell line as a reference for estimating unknown oxygen levels in other cell lines and accounts for high degrees of variability between different cell lines.