Nascimento Jaclyn M, Shi Linda Z, Chandsawangbhuwana Charlie, Tam James, Durrant Barbara, Botvinick Elliot L, Berns Michael W
University of California, San Diego, Department of Electrical and Computer Engineering, 9500 Gilman Drive, La Jolla, California 92093, USA.
J Biomed Opt. 2008 Jan-Feb;13(1):014002. doi: 10.1117/1.2839051.
We combine laser tweezers with custom computer tracking software and robotics to analyze the motility [swimming speed, VCL (curvilinear velocity), and swimming force in terms of escape laser power (Pesc)] and energetics [mitochondrial membrane potential (MP)] of individual sperm. Domestic dog sperm are labeled with a cationic fluorescent probe, DiOC2(3), that reports the MP across the inner membrane of the mitochondria located in the sperm's midpiece. Individual sperm are tracked to calculate VCL. Pesc is measured by reducing the laser power after the sperm is trapped using laser tweezers until the sperm is capable of escaping the trap. The MP is measured every second over a 5-s interval during the tracking phase (sperm is swimming freely) and continuously during the trapping phase. The effect of the fluorescent probe on sperm motility is addressed. The sensitivity of the probe is measured by assessing the effects of a mitochondrial uncoupling agent (CCCP) on MP of free swimming sperm. The effects of prolonged exposed to the laser tweezers on VCL and MP are analyzed. The system's capabilities are demonstrated by measuring VCL, Pesc, and MP simultaneously for individual sperm. This combination of imaging tools is useful to quantitatively assess sperm quality and viability.
我们将激光镊子与定制的计算机跟踪软件及机器人技术相结合，以分析单个精子的运动能力[游泳速度、曲线速度（VCL）以及以逃逸激光功率（Pesc）表示的游泳力]和能量学[线粒体膜电位（MP）]。家犬精子用阳离子荧光探针DiOC2(3)进行标记，该探针可报告位于精子中段的线粒体内膜上的MP。跟踪单个精子以计算VCL。通过在精子被激光镊子捕获后降低激光功率直至精子能够逃离捕获器来测量Pesc。在跟踪阶段（精子自由游动）的5秒间隔内每秒测量一次MP，在捕获阶段则持续测量。研究了荧光探针对精子运动能力的影响。通过评估线粒体解偶联剂（CCCP）对自由游动精子MP的影响来测量探针的灵敏度。分析了长时间暴露于激光镊子对VCL和MP的影响。通过同时测量单个精子的VCL、Pesc和MP来展示该系统的能力。这种成像工具的组合有助于定量评估精子质量和活力。
