Dataset from "Channelrhodopsin-2 Oligomerization in Cell Membrane Revealed by Photo-Activated Localization Microscopy" (CD28-mEos3.2)

This dataset contains raw microscopy data from the article "Channelrhodopsin-2 Oligomerization in Cell Membrane Revealed by Photo-Activated Localization Microscopy" (doi.org/10.1002/anie.202307555) Zip file contains PALM measurements of HEK293 cells expressing CD28 fused with mEos3.2. Each folder in .zip file contains one PALM measurement (TIFF format) and recording settings (.xml) automatically created by acquiring software. For technical reasons, single measurements are divided into TIFF files of =<4088 frames. Detailed protocols for sample preparation and data acquisition are described in the article. Some details are listed below. SAMPLE PREPARATION cell line: Flp-In™ T-REx™ 293 transfection method: modified calcium-phosphate transient transfection [1] fixation method: 4% paraformaldehyde solution in PBS for 30 min at room temperature imaging buffer: PBS PALM ACQUISITION A custom-built setup for single-molecule localization microscopy is described in [2]. mEos3.2 was simultaneously photoconverted, imaged and photobleached by gradually increasing UV illumination (405 nm; up to 1 mW at the sample) and continuous excitation at 561 nm (approximately 50 mW at the sample). PALM movies of cell plasma membrane were recorded in total internal reflection fluorescence (TIRF) mode using an EMCCD camera: exposure time = 100 ms frame size = 512x512 pixels pixel size = 80 nm Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Molecular and cellular biology. 1987 Aug 1;7(8):2745-52. Tang Y, Dai L, Zhang X, Li J, Hendriks J, Fan X, Gruteser N, Meisenberg A, Baumann A, Katranidis A, Gensch T. SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy. Scientific reports. 2015 Jun 22;5(1):11073

本数据集包含发表于论文《Channelrhodopsin-2 Oligomerization in Cell Membrane Revealed by Photo-Activated Localization Microscopy》（DOI：10.1002/anie.202307555）的原始显微成像数据。压缩包内含表达CD28与mEos3.2融合蛋白的HEK293细胞的光激活定位显微镜（Photo-Activated Localization Microscopy，PALM）测量数据。压缩包内每个文件夹对应一次PALM测量结果，包含TIFF格式的成像文件与采集软件自动生成的配置参数XML文件。受技术限制，单次测量的成像文件被分割为帧数不超过4088的TIFF子文件。样本制备与数据采集的详细流程已在论文中阐述，部分关键细节如下：

### 样本制备
- 细胞系：Flp-In™ T-REx™ 293细胞
- 转染方法：改良磷酸钙法瞬时转染[1]
- 固定方法：采用磷酸盐缓冲液（PBS）配制的4%多聚甲醛溶液，室温固定30分钟
- 成像缓冲液：磷酸盐缓冲液（PBS）

### PALM采集
本研究使用的单分子定位显微镜定制化搭建方案已在文献[2]中详述。通过逐步增强405 nm紫外光照（样品处最高光功率达1 mW）并持续以561 nm激光激发（样品处光功率约50 mW），实现对mEos3.2的同步光转换、成像与光漂白。采用全内反射荧光（Total Internal Reflection Fluorescence，TIRF）模式采集细胞质膜的PALM序列图像，使用电子倍增电荷耦合器件（Electron Multiplying Charge-Coupled Device，EMCCD）相机进行记录，相关采集参数如下：
- 曝光时长：100 ms
- 帧尺寸：512×512像素
- 像素尺寸：80 nm

### 参考文献
[1] Chen C, Okayama H. 基于质粒DNA的哺乳动物细胞高效转化. 《分子与细胞生物学》. 1987年8月1日;7(8):2745-52.
[2] Tang Y, Dai L, Zhang X, Li J, Hendriks J, Fan X, Gruteser N, Meisenberg A, Baumann A, Katranidis A, Gensch T. SNSMIL：一种用于超分辨荧光显微镜的实时单分子识别与定位算法. 《科学报告》. 2015年6月22日;5(1):11073