Gliding motility assay using 16551mM D2O in solution. Replicate 1/3. ROI 4/15.

• This is a gliding motility assay using kinesin-1 and rhodamine labeled microtubules. • The buffer used was PEM (also known as BRB80) with the inclusion of 16551mM D2O. • Images were taken with an Andor Luca EMCCD camera on an Olympus IX-71 microscope using a 60x 1.42NA PlanApo objective. • This fileset is replicate 1/3 and ROI 4/15. • Images were taken to investigate speed variations of microtubules due to the inclusion of D2O in the motility assay. • The temperature of the slide was held constant at 32°C by an objective heater. Early ROIs, i.e. 1-5, have slower giding speeds due to the increasing temperature of the slide which, was prepared at room temperature before observation. After ROI 5 the gliding speed stabilizes. • Pixels lengths were measured to be 166nm/pixel using a stage micrometer. • After the files have downloaded, a simple command line entry will convert the images to a movie. Mencoder must be installed. ls > list.txt | mencoder mf://@list.txt -mf w=496:h=658:fps=30:type=png -ovc lavc -lavcopts vcodec=mpeg4:mbd=2:trell -o output.avi • Upload of these files were made possible through the figshare API.• 本实验为基于驱动蛋白-1（kinesin-1）与罗丹明标记微管的滑行运动测定实验。 • 实验所用缓冲液为PEM（亦称BRB80缓冲液），其中添加了16551mM重水（D₂O）。 • 成像环节采用奥林巴斯IX-71型显微镜，搭配Andor Luca EMCCD相机与60× 1.42NA PlanApo物镜完成图像采集。 • 本次文件集对应3组重复实验中的第1组，以及15个感兴趣区域（Region of Interest，ROI）中的第4个。 • 本实验旨在探究滑行运动测定体系中添加重水后，微管滑行速度的变化情况。 • 实验通过物镜加热器将载玻片温度恒定维持在32℃。早期感兴趣区域（即第1~5组）的微管滑行速度较慢，这是由于载玻片于观测前在室温下制备，随着拍摄进程推进温度逐渐升高；待第5组感兴趣区域拍摄完成后，微管滑行速度趋于稳定。 • 通过载物台测微尺（stage micrometer）标定，像素尺寸为166nm/像素。 • 文件下载完成后，可通过简易命令行指令将图像批量转换为视频。需预先安装Mencoder软件，具体指令为：ls > list.txt | mencoder mf://@list.txt -mf w=496:h=658:fps=30:type=png -ovc lavc -lavcopts vcodec=mpeg4:mbd=2:trell -o output.avi • 本次文件上传借助figshare API接口完成。