Jose Elias Do Elias Do Nascimento

Optical trapping is a noninvasive biophotonic tool that has been developed to study the physiological and biomechanical properties of cells. The custom-designed optical system is built to direct near-infrared laser light into an inverted microscope to create a single-point three-dimensional gradient laser trap at the microscope focal point. A real-time automated tracking and trapping system is described that provides a remote user-friendly robotic interface. The combination of laser tweezers, fluorescent imaging, and RATTS can measure sperm swimming speed and swimming force simultaneously with mitochondrial membrane potential. The roles of two sources of adenosine triphosphate in sperm motility/energetics are studied: oxidative phosphorylation, which occurs in the mitochondria located in the spermmidpiece, and glycolysis, which occurs along the length of the sperm tail. The effects of glucose, oxidative phosphorylation inhibitors, and glycolytic inhibitors on human spermmotility are studied. This combination of photonic 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 summary, engineering and biological systems are combined to provide a powerful interdisciplinary approach to study the complex biological systems that drive the sperm toward the egg. © 2010 John Wiley & Sons, Inc.

Sperm cells from a domestic dog were treated with oxacarbocyanine DiOC2, a ratiometrically-encoded membrane potential fluorescent probe in order to monitor the mitochondria stored in an individual sperm's midpiece. This dye normally emits a red fluorescence near 610 nm as well as a green fluorescence near 515 nm. The ratio of red to green fluorescence provides a substantially accurate and precise measurement of sperm midpiece membrane potential. A two-level computer system has been developed to quantify the motility and energetics of sperm using video rate tracking, automated laser trapping and fluorescent imaging. The communication between these two systems is achieved by a networked gigabit TCP/IP cat5e crossover connection. This allows for the curvilinear velocity and ratio of the red to green fluorescent images of individual sperm to be written to the hard drive at video rates. This two-level automatic system has increased experimental throughput over our previous single-level system by an order of magnitude.

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.