Laser scanning microscopy in enzyme histochemistry. Visualization of cerium-based and dab-based primary reaction products of phosphatases, oxidases and peroxidases by reflectance and transmission laser scanning microscopy.
Laser scanning microscopy in enzyme histochemistry. Visualization of cerium-based and dab-based primary reaction products of phosphatases, oxidases and peroxidases by reflectance and transmission laser scanning microscopy.
复制标题
酶组织化学中的激光扫描显微镜。
DOI：
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发表时间：
1998
影响因子：
1.6
通讯作者：
K. König
中科院分区：
文献类型：
--
作者：
K. Halbhuber;R. Krieg;K. König
共聚焦激光扫描显微镜的反射模式适用于检测氧化酶（CeIV-过氧化氢）和磷酸酶（CeIII-羟基磷酸盐转化为CeIV-过羟基磷酸盐）的铈基初级反应产物，以及细胞色素C氧化酶和过氧化物酶在振动切片机、冷冻切片机和半薄塑料切片中的DAB基初级反应产物（Ni-DAB、Ni-FeII-DAB和CeIV-DAB复合物）。与共焦检测相结合，可以获得具有亚微米空间分辨率的3D图像。此外，CeIV-过氢氧化物、CeIV-过羟基磷酸盐、CeIV-DAB复合物和儿茶酚-DAB聚合物具有高吸收性。在其他添加剂中，特别是稳定的硝酰基自由基导致DAB染色在灵敏度和适当定位方面的明显改善。另外，在几个实验中，通过辣根过氧化物酶稀释系列的印迹证明了这一点。在切片中，可以容易地记录在激发氩离子激光的波长（优选488 nm）下的反射率信号和高透射对比度。含铈的模型沉淀物的吸胀研究的结果表明，铈一般应被氧化前观察，因为CeIV化合物的折射率是相当高的比相应的CeIII化合物。在形态相似的部分，从反射部分的反射强度的比较数值评估是可能的。共聚焦激光扫描显微镜提供了一种独特的方式，高分辨率检测的主要组织化学反应产物是充分反射和/或吸收。所提出的技术可能为基础研究和医学诊断开辟新的方法学可能性。
The reflectance mode of confocal laser scanning microscopy is suitable to detect cerium-based primary reaction products of oxidases (CeIV-perhydroxide) and phosphatases (CeIII-hydroxy-phosphate converted into CeIV-perhydroxy-phosphate) as well as of DAB-based primary reaction products (Ni-DAB, Ni-FeII-DAB and CeIV-DAB complexes) of cytochrome C oxidase and peroxidases in vibratome, cryotome and semithin plastic sections. In combination with confocal detection 3D images with submicron spatial resolution can be obtained. Moreover, CeIV-perhydroxide, CeIV-perhydroxy-phosphate, CeIV-DAB complexes and catechol-DAB polymers are highly absorptive. Among other additives, especially stable nitroxyl radicals led to a distinct improvement of the DAB staining in terms of sensitivity and proper localization. This was proven in addition by means of blotting a horseradish peroxidase dilution series during several experiments. In sections it was easily possible to record reflectance signals and high transmission contrast at the wavelength of the exciting argon ion laser (preferentially 488 nm). The results of an imbibition study of cerium-containing model precipitates indicate that the cerium generally should be oxidized prior to observation because the index of refraction of CeIV compounds is considerably higher than that of the corresponding CeIII compounds. A comparative numerical assessment of reflection intensities from reflectant parts in morphologically similar sections is possible. Confocal laser scanning microscopy offers a unique way for high resolution detection of primary histochemical reaction products being sufficiently reflective and/or absorptive. The proposed techniques may open new methodological possibilities for basic research and for medical diagnosis.