Non-invasive manipulation and analysis of biological objects with high resolution and efficiency have become extremely important. This dissertation presents three novel techniques based on light scattering and optical forces, which could bring high resolution and speed to submicron cell characterization, improve the throughput and functionality of self-propelled cell analysis and enhance the parallelism, portability and flexibility of cell manipulation instruments. Elastic light scattering is used for submicron cell characterization. An important problem in oceanic microbial ecology is characterizing the constituents of the sea. To pursue this goal, the application of angularly-dependent light scattering on oceanic microbe differentiation has been explored. Good overall agreement is found between scattering patterns simulated with developed models and those experimentally measured. The distinct scattering patterns of different species provide fingerprint information that will allow for routine identification of marine picoplankton. Optical tweezers have been used not only for manipulating cells, viruses and organelles within cells, but also measuring biological forces on the order of picoNewtons. In the second part of this dissertation, a three-dimensional resizable annular laser trap is developed for self- propelled cell manipulation and analysis. This system offers high power efficiency and is potentially useful for high-throughput multi-level sperm sorting based on motility and chemotaxis. With only tens of milliwatts devoted to each sperm, this new type of laser trap offers a gentle way to study the effect of optical force, laser radiation and external obstacles on sperm swimming patterns and membrane potential in detail. Applications could be extended to motility and biotropism studies on other self-propelled cells, such as algae and bacteria, etc. The third part of this dissertation involves manipulation of multiple biological cells both synchronously and independently. Substituting Vertical Cavity Surface Emitting Lasers (VCSELs) for standard diode and gas lasers in optical micromanipulation provides the ability to meet the miniaturization and parallelism demands of current lab-on-a-chip technologies, so that multiple experiments can be performed in parallel and at low costs. By combining a single VCSEL trap that can move individual particles in 3-D with a VCSEL trap array, a micromanipulator capable of multi-step group operating and cell rotating is constructed

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.

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.

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 (single sperm per spot), lacks the ability of in-situ sorting based on motility and chemotaxis, requires high laser power (hundreds of milliWatts) 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 (tens to hundred sperm per ring) 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.

As a powerful and noninvasive tool, laser trapping has been widely applied for the confinement and physiological study of biological cells and organelles. Researchers have used the single spot laser trap to hold individual sperm and quantitatively evaluated the motile force generated by a sperm. Early studies revealed the relationship between sperm motility and swimming behavior and helped the investigations in medical aspects of sperm activity. As sperm chemotaxis draws more and more interest in fertilization research, the studies on sperm-egg communication may help to explain male or female infertility and provide exciting new approaches to contraception. However, single spot laser trapping can only be used to investigate an individual target, which has limits in efficiency and throughput. To study the chemotactic response of sperm to eggs and to characterize sperm motility, an annular laser trap with a diameter of several hundred microns is designed, simulated with ray tracing tool, and implemented. An axicon transforms the wavefront such that the laser beam is incident on the microscope objective from all directions while filling the back aperture completely for high efficiency trapping. A trapping experiment with microspheres is carried out to evaluate the system performance. The power requirement for annular sperm trapping is determined experimentally and compared with theoretical calculations. With a chemo-attractant located in the center and sperm approaching from all directions, the annular laser trapping could serve as a speed bump for sperm so that motility characterization and fertility sorting can be performed efficiently.

A diameter-adjustable annular laser trapping system based on axicons was designed for sperm chemotactaxis and fertilizability characterization. Experiment on microspheres testified the feasibility and performance of the system and was consistent with theoretical expectation. © 2005 Optical Society of America.