Use of laser tweezers to analyze sperm motility and mitochondrial membrane potential

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, DiOC23, 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 twee- zers until the sperm is capable of escaping the trap. The MP is mea- sured 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 ad- dressed. 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 twee- zers on VCL and MP are analyzed. The system's capabilities are dem- onstrated by measuring VCL, Pesc, and MP simultaneously for indi- vidual sperm. This combination of imaging tools is useful to quantitatively assess sperm quality and viability. © 2008 Society of Photo-