Juliana Nascimento

We have developed a microscope system for real-time single sperm tracking with an automated laser tweezers escape power assay. Phase contrast images of swimming sperm are digitized to the computer at video rate. The custom algorithm creates a region of interest centered about a sperm in response to a mouse click and performs all subsequent tasks autonomously. Microscope stage movement responds to feedback from video analysis of swimming sperm to center the sperm with respect to the field of view. For escape power assays, sperm are automatically relocated to the laser trap focus where they are held for a user-defined duration at fixed power, or held as laser power is gradually reduced. The sperm's position is automatically monitored to measure the laser power at which the sperm escapes the trap. Sperm are tracked for extended durations before and after laser trap experiments. Motility measurements including the curvilinear velocity and the absolute position of the sperm relative to the cell chamber are calculated and written to the hard drive at video rate. Experimental throughput is increased over 30 times compared to off-line data analysis. The efficacy of the "track and trap" algorithm is validated through examples and comparisons with the manually collected data. © 2006 Wiley-Liss, Inc.

The combination of laser tweezers, fluorescent imaging, and real-time automated tracking and trapping can measure sperm swimming speed and swimming force simultaneously with mitochondrial membrane potential. This approach is used to study the roles of two sources of ATP in sperm motility: oxidative phosphorylation, which occurs in the mitochondria located in the sperm midpiece and glycolysis, which occurs along the length of the sperm tail. The relationships between swimming speed and MMP and swimming force and MMP are studied in dog and human sperm. The effects of glucose, oxidative phosphorylation inhibitors and glycolytic inhibitors on human sperm motility are examined. The results indicate that oxidative phosphorylation does contribute some ATP for human sperm motility, but not enough to sustain high motility. The glycolytic pathway is shown to be a primary source of energy for human sperm motility. © 2008 Wiley-Liss, Inc.

Optical trapping is a non-invasive biophysical tool which has been widely applied to study physiological and biomechanical properties of cells. Using laser 'tweezers' in combination with custom-designed computer tracking algorithms, the swimming speeds and the relative swimming forces of individual sperm can be measured in real time. This combination of physical and engineering tools has been used to examine the evolutionary effect of sperm competition in primates. The results demonstrate a correlation between mating type and sperm motility: sperm from polygamous primate species swim faster and with greater force than sperm from polygynous primate species. In addition, sperm swimming force linearly increases with swimming speed for each species, yet the regression relating the two parameters is species specific. These results demonstrate the feasibility of using these tools to study rapidly moving biological cells. © 2007 The Royal Society.

Sperm motility is an important concept in fertility research. To this end, single spot laser tweezers have been used to quantitatively analyze the motility of individual sperm. However, this method is limited with throughput, lacks the ability of in-situ sorting based on motility and chemotaxis, requires high laser power and can not be used to dynamically monitor changes in sperm swimming behavior under the influence of a laser beam. Here, we report a continuous 3-D ring-shaped laser trap which could be used for multi-level and high-throughput sperm sorting based on their motility and chemotaxis. Under a laser power of only tens of milliWatts, human sperm with low to medium velocity are slowed down, stopped, or forced to change their trajectories to swim along the ring due to the optical gradient force in the radial direction. This is the first demonstration of parallel sperm sorting based on motility with optical trapping technology. In addition, by making the sperm swimming along the circumference of the ring, the effect of laser radiation, optical force and external obstacles on sperm energetics are investigated in a more gentle and quantitative way. The application of this method could be extended to motility and bio-tropism studies of other self-propelled cells, such as algae and bacteria. © Springer Science+Business Media, LLC 2007.

To study the chemotactic response of sperm to an egg and to characterize sperm motility, an annular laser trap based on axicons is designed, simulated with the ray-tracing tool, and implemented. The diameter of the trapping ring can be adjusted dynamically for a range of over 400 μm by simply translating one axicon along the optical axis. Trapping experiments with microspheres and dog sperm demonstrate the feasibility of the system, and the power requirement agrees with theoretical expectation. This new type of laser trapping could provide a prototype of a parallel, objective, and quantitative tool for animal fertility and biotropism study. © 2006 Optical Society of America.