FULL ARTICLE Automatic annular laser trapping: a system for high-throughput sperm analysis and sorting Linda Shi * ; 1 Bing Shao 2 , Timothy Chen, 1 and Michael Berns 2 1 UCSD – Bioengineering, 9500 Gilman Dr. SERF 295, MC 0435 La Jolla California 92093, USA 2 UCI – Beckman Laser Institute, 1002 Health Sciences Road, Irvine, CA 92612, USA Received 4 September 2008, revised 20 October 2008, accepted 23 October 2008 Published online 5 December 2008 Key words: real-time sperm tracking, automatic annular laser trap, sperm sorting, sperm motility # 2009 by WILEY-VCH Verlag GmbH& Co. KGaA, Weinheim 1. Introduction Real-time computer-based tracking of live cells has become an important tool in biology. A robotic image- guided real-time respiratory tumor tracking system was discussed in Muacevic et al. 2007 [1]. The respira- tory tracking error stated in [1] was less than 1 mm and the overall shape of the dose profile was not af- fected by target motion and/or phase shift between fi- ducial and optical marker motion. A real-time motion tracking algorithm was applied to ultrasonic strain imaging in Jiang & Hall, 2007 [2]. The modified pre- dictive search block-matching algorithm in [2] can not only obtain high contrast strain images, but also reach 10 Hz frame rates. A real-time image-guided direct convective perfusion of intrinsic brainstem lesions was demonstrated by Lonser et al., 2007 [3]. It demon- strated that convection-enhanced delivery (CED) can be used to perfuse the brain and brainstem with thera- peutic agents while simultaneously tracking their dis- tribution using coinfusion of a surrogate magnetic re- sonance (MR) imaging tracer [3]. In the area of live cell tracking, sperm cells are one of the most difficult cell types due to their fast swimming speed (up to # 2009 by WILEY-VCH Verlag GmbH& Co. KGaA, Weinheim Journal of BIOPHOTONICS An automatic microscope system is designed to study the response of sperm motility to an annular laser trap. A continuous annular laser trap provides a parallel way to analyze and sort sperm based on their motility and to study the effects of laser radiation, optical force and ex- ternal obstacles. In the described automatic microscope system, the phase contrast images of swimming sperm are digitized to the computer at video rates. The micro- scope stage is controlled in real-time to relocate the sperm of interest to the annular trap with a normal or tangential entering angle. The sperm is continuously tracked and the swimming behavior is identified. Using this system, parallel sorting on human and gorilla sperm are achieved and threshold power levels separating the “fast” group and the “slow” group are compared for those two species. Phase contract image of sperm moving under the micro- scope captured in real-time. The bright dot is a moving sperm. The green box is the region of interest created. The big yellow circle indicates the annular trap location. * Corresponding author: e-mail: zhixiashi@gmail.com J. Biophoton. 2, No. 3, 167–177 (2009) / DOI 10.1002/jbio.200810053