Objective and quantitative assessment of sperm quality is important for human infertility as well as for animal husbandry. Several techniques, including subjectively scoring sperm based on speed of progression and quantitatively measuring sperm motility parameters, are currently used to assess sperm. Optical tweezers provide a non-invasive way to study sperm motility by measuring sperm swimming forces. The minimum amount of laser power needed to hold the sperm in the trap (or the threshold escape power) is directly proportional to the sperm's swimming force (F = Q * P / c, where F is the swimming force, P is the laser power, c is the speed of light in the medium, and Q is the geometrically determined trapping efficiency parameter). The purpose of this dissertation is to first develop an objective and quantitative method to analyze sperm motility based on sperm swimming force. A custom system is developed to track, trap, and fluorescently image sperm, measuring sperm swimming speed, swimming force and mitochondrial membrane potential in real-time. This system is then used to study sperm. Specifically, the effects of trap duration and laser trapping power on sperm motility are determined, the relationships between sperm swimming force and swimming speed for various mammalian species are defined, both the effects of cryopreservation and sperm competition in primate species on motility are studied, and finally the relationship between sperm motility (swimming force and swimming speed) and energy production is analyzed

This study examines the use of optical trapping as a quantitative measure of sperm motility. The effects of laser trap duration and laser trapping power on sperm motility are described between sperm swimming force, swimmimg speed, and speed of progression (SOP) score. Sperm (SOP scores of 2-4) were trapped by a continuous-wave 1064 nm single-point gradient laser trap. Trap duration effects were quantified for 15, 10, and 5 seconds at 420 mW laser power. Laser power effects were quantified at powers of 420 mW, 350 mW, 300 mW, and 250 mW for five seconds. Swimming force, swimming speed, and SOP score relationships were examined at a trap duration and trapping power shown to minimally affect sperm motility. Swimming forces were measured by trapping sperm and subsequently decreasing laser power until the sperm escaped the trap. Swimming trajectories were calculated by custom-built software, and SOP scores were assigned by three qualified sperm scoring experts. A ubiquitous class of sperm were identified that swim with relatively high forces that are uncorrelated to swimming speed. It is concluded that sperm swimming forces measured by optical trapping provide new and valuable quantitative information to assess sperm motility.

This paper describes a robust single sperm tracking algorithm (SSTA) that can be used in laser optical trapping and sperm motility studies. The algorithm creates a region of interest (ROI) centered about a sperm selected by the user. SSTA contrast enhances the ROI image and implements a modified four-class thresholding method to extract the tracked sperm as it transitions in and out of focus. The nearest neighbor method is complemented with a speed-check feature to aid tracking in the presence of additional sperm or other particles. SSTA has a collision-detection feature for real or perceived collision or near-miss cases between two sperm. Subsequent postcollision analysis employs three criteria to distinguish the tracked sperm in the image. The efficacy of SSTA is validated through examples and comparisons to commercially available computer-aided sperm tracking systems.

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 (RATTS) 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 (MMP). The roles of two sources of adenosine triphosphate in sperm motility/energetics are studied: oxidative phosphorylation, which occurs in the mitochondria located in the sperm midpiece, and glycolysis, which occurs along the length of the sperm tail (flagellum). The effects of glucose, oxidative phosphorylation inhibitors, and glycolytic inhibitors on human sperm motility 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 (multi-partner) primate species swim faster and with greater force than sperm from polygynous (single partner) 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.

A three-dimensional (3D) ring-shaped laser trap has been built using axicons. The diameter of this laser trap ranges from 70 to 140 mum and is adjusted by simply changing the position of one axicon in the optical path. Parallel 3D trapping of 5 mum silica microspheres and 3D confinement of cells along the ring are demonstrated. In this system the special optical properties of axicons are used to create a continuous annular trap with high power efficiency and a constant numerical aperture. This new approach, without any mechanical scanning, offers significant potential for applications in cell motility analysis and biotropism studies.

An integrated robotic laser and microscope system has been developed to automatically analyze individual sperm motility and energetics. The custom-designed optical system directs near-infrared laser light into an inverted microscope to create a single-point 3-D gradient laser trap at the focal spot of the microscope objective. A two-level computer structure is described that quantifies the sperm motility (in terms of swimming speed and swimming force) and energetics (measuring mid-piece membrane potential) using real-time tracking (done by the upper-level system) and fluorescent ratio imaging (done by the lower-level system). The communication between these two systems is achieved by a gigabit network. The custom-built image processing algorithm identifies the sperm swimming trajectory in real-time using phase contrast images, and then subsequently traps the sperm by automatically moving the microscope stage to relocate the sperm to the laser trap focal plane. Once the sperm is stably trapped (determined by the algorithm), the algorithm can also gradually reduce the laser power by rotating the polarizer in the laser path to measure the trapping power at which the sperm is capable of escaping the trap. To monitor the membrane potential of the mitochondria located in a sperm's mid-piece, the sperm is treated with a ratiometrically-encoded fluorescent probe. The proposed algorithm can relocate the sperm to the center of the ratio imaging camera and the average ratio value can be measured in real-time. The three parameters, sperm escape power, sperm swimming speed and ratio values of the mid-piece membrane potential of individual sperm can be compared with respect to time. This two-level automatic system to study individual sperm motility and energetics has not only increased experimental throughput by an order of magnitude but also has allowed us to monitor sperm energetics prior to and after exposure to the laser trap. This system should have application in both the human fertility clinic and in animal husbandry.

Laser trapping in the near infrared regime is a noninvasive and microfluidic-compatible biomedical tool. This chapter examines the use of optical trapping as a quantitative measure of sperm motility. The single point gradient trap is used to directly measure the swimming forces of sperm from several different species. These forces could provide useful information about the overall sperm motility and semen quality. The swimming force is measured by trapping sperm and subsequently decreasing laser power until the sperm is capable of escaping the trap. Swimming trajectories were calculated by custom built software, an automatic sperm tracking algorithm called the single sperm tracking algorithm or SSTA. A real-time automated tracking and trapping system, or RATTS, which operates at video rate, was developed to perform experiments with minimal human involvement. After the experimenter initially identifies and clicks the computer mouse on the sperm-of-interest, RATTS performs all further tracking and trapping functions without human intervention. Additionally, an annular laser trap which is potentially useful for high-throughput sperm sorting based on motility and chemotaxis was developed. This low power trap offers a more gentle way for studying the effects of laser radiation, optical force, and external obstacles on sperm swimming pattern.

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 microm 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.

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.