Photoconversion of Hoechst

The data set provided is a time series of image stacks showing the 405 nm laser-induced emission change of the DNA intercalating dye Hoechst from blue to green in living U2OS cells.<br> <br>It is part of the following research note:<br>Hurst V and Gasser SM. The study of protein recruitment to laser-induced DNA lesions can be distorted byphotoconversion of the DNA binding dye Hoechst. F1000Research , :104. <br>Experimental details:<br>U2OS cells (a gift from Prof. Primo Leo Schaer, Department of Biomedicine, University of Basel) were incubated with 1.5 µg/ml Hoechst 33342 (Thermo Fisher Scientific, H1399) for at least 30 minutes prior to photoconversion. Photoconversion was induced with a VisiFRAP module (Visitron) mounted on the backport of the microscope and equipped with a 405 nm laser (Toptica, illumination power at the objective 12.8 mW). Confocal images were acquired with an Olympus IX81 microscope equipped with a PlanApo 100x/1.45 TIRFM oil objective, a CSU-X1 scan-head (Yokogawa), an Evolve 512 EMCCD camera (Photometrics), a 491nm laser (Cobolt Calypso 100), a 488/568 dichroic (Semrock Di01-T488/568-13x15x0.5), a band-pass 525/40 emission filter (Semrock FF01-525/40-25) and controlled with the Visiview Software (Visitron).<br>

本数据集为一组时序图像栈，展示了活U2OS细胞内DNA嵌入染料赫斯特染料（Hoechst）在405 nm激光诱导下从蓝色到绿色的发射信号变化。

本数据集隶属于以下研究简报：
赫斯特V（Hurst V）与加瑟SM（Gasser SM）. 激光诱导DNA结合染料赫斯特染料的光转化可干扰蛋白质向激光诱导DNA损伤位点的募集研究. 《F1000Research》，104.

实验细节如下：
U2OS细胞（由巴塞尔大学生物医学系普里莫·莱奥·沙尔教授惠赠）在光转化前，以1.5 μg/ml的赫斯特33342（Hoechst 33342，赛默飞世尔科技，货号H1399）孵育至少30分钟。光转化过程通过安装于显微镜后端口的VisiFRAP模块（Visitron公司）完成，该模块搭载405 nm激光器（德国Toptica公司，物镜处照明功率为12.8 mW）。
共聚焦图像采集使用奥林巴斯IX81显微镜完成，该显微镜搭载PlanApo 100×/1.45 全内反射荧光显微镜（TIRFM）油浸物镜、横河电机（Yokogawa）CSU-X1扫描头、Evolve 512 电子倍增电荷耦合器件（EMCCD）相机（Photometrics公司）、491 nm激光器（Cobolt Calypso 100）、488/568二向色镜（Semrock Di01-T488/568-13×15×0.5）以及525/40带通发射滤光片（Semrock FF01-525/40-25），并通过Visiview软件（Visitron公司）控制。