Cell extraction automation in single cell surgery using the displacement method.

Micromanipulation is the precise in vitro handling and study of individual biological cells, where the smallest error can be disastrous. One such example is the extraction of cellular material from multicellular organisms, such as cells from early stage embryos. In this paper, we propose automation methods for the extraction and retrieval of individual cells from a multicellular organism in vitro using the displacement method. Computer-controlled syringe pumps and micromanipulators combined with custom computer vision algorithms are used for automated cell extraction and retrieval. Automation feasibility is demonstrated through automated controlled extraction of one or two blastomeres from cleavage-stage embryos. Preliminary proof of concept blastomere extraction experiments involving mouse embryos obtained success rates ranging from 72% to 88% for the different extraction tasks: displacement, detection, and retrieval. These automated blastomere extraction experiments demonstrate that automated cell extraction is indeed feasible, but the process may still be improved. To the best of these authors' knowledge, this paper is the first to report the automation of single cell extraction from multicellular organisms using the displacement method, and especially for automated blastomere extraction from cleavage-stage embryos. These methods provide a set of tools for moving towards fully automated single cell surgery procedures.

Automation and Optimization of Multipulse Laser Zona Drilling of Mouse Embryos During Embryo Biopsy.

Laser zona drilling (LZD) is a required step in many embryonic surgical procedures, for example, assisted hatching and preimplantation genetic diagnosis. LZD involves the ablation of the zona pellucida (ZP) using a laser while minimizing potentially harmful thermal effects on critical internal cell structures. OBJECTIVE: Develop a method for the automation and optimization of multipulse LZD, applied to cleavage-stage embryos. METHODS: A two-stage optimization is used. The first stage uses computer vision algorithms to identify embryonic structures and determines the optimal ablation zone farthest away from critical structures such as blastomeres. The second stage combines a genetic algorithm with a previously reported thermal analysis of LZD to optimize the combination of laser pulse locations and pulse durations. The goal is to minimize the peak temperature experienced by the blastomeres while creating the desired opening in the ZP. RESULTS: A proof of concept of the proposed LZD automation and optimization method is demonstrated through experiments on mouse embryos with positive results, as adequately sized openings are created. CONCLUSION: Automation of LZD is feasible and is a viable step toward the automation of embryo biopsy procedures. SIGNIFICANCE: LZD is a common but delicate procedure performed by human operators using subjective methods to gauge proper LZD procedure. Automation of LZD removes human error to increase the success rate of LZD. Although the proposed methods are developed for cleavage-stage embryos, the same methods may be applied to most types LZD procedures, embryos at different developmental stages, or nonembryonic cells.

Multi-Pulse laser ablation modeling with applications to automated zona removal.

Laser zona drilling (LZD), the ablation of a portion of the zona pellucida (ZP) in embryos with the use of a laser, is a required step in many embryonic surgical procedures such as assisted hatching and preimplantation genetic diagnosis. The objective of LZD is to remove specific locations of the ZP while minimizing potential harmful thermal effects to important structures of the embryo, namely the blastomeres. Current thermal analyzes of lasers used in LZD only encompass the use of a single pulse, whereas LZD is typically performed using multiple pulses. In this paper we analyze the effect of multipulse LZD and introduce a linear approximation method for multi-pulse LZD. Furthermore, we describe a novel method of measuring the thermal effect of a single laser pulse using the thermosensitive fluorescent dye Rhodamine B and a high speed camera.