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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Multilayer Three-dimensional Super Resolution Imaging Of Thick Biological Samples
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Multilayer Three-dimensional Super Resolution Imaging Of Thick Biological Samples

机译：厚生物样品的多层三维超分辨率成像

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摘要

Recent advances in optical microscopy have enabled biological imaging beyond the diffraction limit at nanometer resolution. A general feature of most of the techniques based on photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM) has been the use of thin biological samples in combination with total internal reflection, thus limiting the imaging depth to a fraction of an optical wavelength. However, to study whole cells or organelles that are typically up to 15 μm deep into the cell, the extension of these methods to a three-dimensional (3D) super resolution technique is required. Here, we report an advance in optical microscopy that enables imaging of protein distributions in cells with a lateral localization precision better than 50 nm at multiple imaging planes deep in biological samples. The approach is based on combining the lateral super resolution provided by PALM with two-photon temporal focusing that provides optical sectioning. We have generated super-resolution images over an axial range of ≈10 μm in both mitochondrially labeled fixed cells, and in the membranes of living S2 Drosophila cells.
机译：光学显微镜的最新进展已使生物成像超出了纳米分辨率的衍射极限。基于光激活定位显微镜（PALM）或随机光学重建显微镜（STORM）的大多数技术的一般特征是结合了全内反射使用薄生物样本，从而将成像深度限制在光学的一小部分波长。但是，要研究通常深入到细胞深达15μm的整个细胞或细胞器，则需要将这些方法扩展到三维（3D）超分辨率技术。在这里，我们报告了光学显微镜技术的进步，该技术能够在生物样品深处的多个成像平面上以优于50 nm的横向定位精度对细胞中的蛋白质分布进行成像。该方法基于将PALM提供的横向超分辨率与提供光学切片的双光子时间聚焦相结合。我们已经在线粒体标记的固定细胞和活的果蝇S2果蝇细胞膜中产生了≈10μm轴向范围的超分辨率图像。

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