Angular Approach Scanning Ion Conductance Microscopy.
Angular Approach Scanning Ion Conductance Microscopy.
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DOI：
10.1016/j.bpj.2016.04.017
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发表时间：
2016-05-24
影响因子：
3.4
通讯作者：
Korchev YE
中科院分区：
文献类型：
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作者：
Shevchuk A;Tokar S;Gopal S;Sanchez-Alonso JL;Tarasov AI;Vélez-Ortega AC;Chiappini C;Rorsman P;Stevens MM;Gorelik J;Frolenkov GI;Klenerman D;Korchev YE
扫描离子电导显微镜(SICM)是一种超分辨率实时成像技术，它使用玻璃纳米管作为成像探针，产生细胞表面的三维(3D)图像。SICM可以用来分析纳米级的细胞形态，跟踪膜动力学，精确地将成像纳米管定位在感兴趣的结构附近，并使用它来获得离子通道记录或局部施加刺激或药物。然而，由于当前可用的SICM系统的局限性，这些SICM优势的实际实现通常是复杂的，这些系统继承了从扫描组件放置在样品正上方的其他扫描探针显微镜的设计。这样的布置使得难以设置相位对比度所需的最佳照明或难以使用高倍率直立光学元件。在这里，我们描述了允许将SICM扫描头安装在标准膜片钳微操作器上并以可调整的接近角度成像样本的设计。这个角度可能和膜片钳移液管在浸水物镜和标本之间的接近角一样浅。使用这种角度方法SICM，我们获得了生长在非透明纳米针阵列上的细胞、朗格汉斯胰岛和直立光学显微镜下的海马神经元的地形图。我们还成像了以前无法接触到的细胞区域，如毛细胞立体纤毛的侧面和分离的心肌细胞的间盘，并从后者进行了有针对性的膜片钳记录。因此，据我们所知，我们新的角度方法SICM允许在不透明的基质上对活细胞进行成像，并在倒置或直立显微镜上与大多数膜片钳设置无缝集成，这将促进细胞生物物理学和生理学的研究。
Scanning ion conductance microscopy (SICM) is a super-resolution live imaging technique that uses a glass nanopipette as an imaging probe to produce three-dimensional (3D) images of cell surface. SICM can be used to analyze cell morphology at nanoscale, follow membrane dynamics, precisely position an imaging nanopipette close to a structure of interest, and use it to obtain ion channel recordings or locally apply stimuli or drugs. Practical implementations of these SICM advantages, however, are often complicated due to the limitations of currently available SICM systems that inherited their design from other scanning probe microscopes in which the scan assembly is placed right above the specimen. Such arrangement makes the setting of optimal illumination necessary for phase contrast or the use of high magnification upright optics difficult. Here, we describe the designs that allow mounting SICM scan head on a standard patch-clamp micromanipulator and imaging the sample at an adjustable approach angle. This angle could be as shallow as the approach angle of a patch-clamp pipette between a water immersion objective and the specimen. Using this angular approach SICM, we obtained topographical images of cells grown on nontransparent nanoneedle arrays, of islets of Langerhans, and of hippocampal neurons under upright optical microscope. We also imaged previously inaccessible areas of cells such as the side surfaces of the hair cell stereocilia and the intercalated disks of isolated cardiac myocytes, and performed targeted patch-clamp recordings from the latter. Thus, our new, to our knowledge, angular approach SICM allows imaging of living cells on nontransparent substrates and a seamless integration with most patch-clamp setups on either inverted or upright microscopes, which would facilitate research in cell biophysics and physiology.