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Distribution of ALA metabolic products in esophageal carcinoma cells using spectrally resolved confocal laser microscopy

机译：使用光谱分辨共聚焦激光显微镜分布ALA代谢产物在食管癌细胞中的分布

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Aminolevulinic acid (ALA) is an efficient substance used in photodynamic therapy (PDT). It is a precursor of light-sensitive products that can selectively accumulate in malignant cells following the altered activity of the heme biosynthetic pathway enzymes in such cells. These products are synthesized in mitochondria and distributed to various cellular structures [1]. The localization of ALA products in subcellular structures depends on their chemical characteristics as well as on the properties of the intracellular environment [2]. Characterization of such properties is possible by means of fluorescent probes like JC-1 and carboxy SNARF-1. However, the emission spectra of these probes are overlapped with spectral pattern of typical ALA product -protoporphyrin IX (PpIX). Spectral overlap of fluorescence signals prevents to clearly separate a distribution of probes from PpIX distribution what can completely mess the applicability of these probes in characterization of cell properties. The spectrally resolved confocal laser microscopy can be used to overcome this problem. In this study, a distribution of ALA metabolic products in relation to the mitochondrial membrane potential and intracellular pH was examined. Human cell lines (KYSE-450, KYSE-70) from esophageal squamous cell carcinoma were used. Cells were incubated with 1mM solution of ALA for four hours. Two fluorescent probes, carboxy SNARF-1 and JC-1 , were used to monitor intracellular pH levels and to determine membrane potential changes, respectively. The samples were scanned by spectrally resolved laser scanning microscope. Spectral linear unmixing method was used to discriminate and separate regions of accumulation of ALA metabolic products of JC-1 and carboxy SNARF-1.
机译：氨基硫酸（ALA）是光动力治疗（PDT）中使用的有效物质。它是光敏产品的前体，其可以在这些细胞中血红素生物合成途径酶的改变活性后选择性地积聚恶性细胞中。这些产品在线粒体中合成，并分布于各种细胞结构[1]。亚细胞结构中ALA产品的定位取决于其化学特性以及细胞内环境的性质[2]。通过荧光探针如JC-1和羧基Snarf-1等荧光探针，可以进行这些性质的表征。然而，这些探针的发射光谱与典型ALA产物的光谱模式重叠 - 促突卟啉IX（PPIX）。荧光信号的光谱重叠可防止从PPIX分布中清楚地分开探针的分布可以完全混淆这些探针在细胞性质表征中的适用性。可用于克服该问题的光谱分辨的共聚焦激光显微镜。在该研究中，检查了与线粒体膜电位和细胞内pH相关的ALA代谢产物的分布。使用来自食道鳞状细胞癌的人细胞系（Kyse-450，Kyse-70）。将细胞与1mM的Ala溶液一起温育4小时。用于监测细胞内pH水平并分别测量细胞内pH水平并分别测定膜电位变化的两种荧光探针。通过光谱分辨的激光扫描显微镜扫描样品。光谱线性解混方法用于区分和分离JC-1和羧基肠杆菌-1的ALA代谢产物的积累区域。

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