TIRF, dSTORM and PREM images supporting data in figure 2 of "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells"

This dataset is part of a collection of imaging data (https://doi.org/10.25444/nhlbi.c.5405490) from the publication entitled: "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells." by Prasai et al. 2021 (https://doi.org/10.1038/s41467-021-24167-9). <br>Figure 2 contains the source of the representative correlative superresolution light and electron microscopy (CLEM) images shown in fig.2a-e. And, it contains all the underlying images that were used in generating fluorescence profiles for dGFP-Rab3a, Rab27a, Rabphilin3a, Granuphilin, and Anti-Rim2 in figure 2h.<br>For CLEM, PC12 cells were were maintained in growth media containing DMEM (Life Technologies), 10% fetal bovine serum (Life Technologies 26140-079), and 1% vol/vol penicillin/streptomycin. They were transiently transfected with DCV marker NPY-mNG and dGFP tagged proteins of interests. Cells were unroofed, fixed, labeled with Alexafluor 647 nanobody, and imaged with Nikon NSTORM microscope for dSTORM. Fluorescently imaged samples were prepared for EM that included fixation in 2% glutaraldehyde, staining with tannic acid and uranyl acetate, serial dehydration with ethanol, critical point drying, metal coating, replica lifting, and imaging with JEOL 1400 equipped with SerialEM for montaging. Processed dSTORM and EM images were correlated using an affine spatial transformation with nearest-neighbor interpolation to map the Gaussian centers of gold nanorods visible in both dSTORM and EM images.

本数据集隶属于一组成像数据集合（https://doi.org/10.25444/nhlbi.c.5405490），其来源为Prasai等人2021年发表的题为《神经内分泌细胞中胞吐致密核心囊泡对接的纳米级分子形态》的研究论文（https://doi.org/10.1038/s41467-021-24167-9）。<br>图2包含了图2a-e中展示的代表性关联超分辨光学与电子显微镜（Correlative Superresolution Light and Electron Microscopy, CLEM）图像的来源，同时包含了用于生成图2h中dGFP-Rab3a、Rab27a、Rabphilin3a、Granuphilin及抗Rim2荧光分布曲线的所有原始图像。<br>在CLEM实验中，PC12细胞培养于含高糖杜尔贝科改良伊格尔培养基（DMEM，Life Technologies）、10%胎牛血清（Life Technologies 26140-079）及1%体积分数青霉素-链霉素的完全培养基中。将致密核心囊泡（Dense Core Vesicle, DCV）标记物NPY-mNG与目标蛋白的dGFP融合载体瞬时转染至细胞内。随后对细胞进行去顶膜处理，固定后用Alexa Fluor 647标记的纳米抗体染色，并使用尼康NSTORM显微镜进行dSTORM成像。对完成荧光成像的样品进行电子显微镜制样：依次经2%戊二醛固定、单宁酸与醋酸铀染色、乙醇梯度脱水、临界点干燥、金属镀膜及复型剥离，最终使用搭载SerialEM软件的JEOL 1400电子显微镜进行拼接成像。通过仿射空间变换结合最近邻插值法，匹配dSTORM与电子显微镜图像中均可观测到的金纳米棒高斯中心，以此完成两类图像的关联配准。