Microscope-delivered ultraviolet laser zona dissection: Principles and practices

Journal of Assisted Reproduction and Genetics, Vol. 9, No. 6, 1992 LASER Microscope-Delivered Ultraviolet Laser Zona Dissection: Principles and Practices INTRODUCTION amounts of water, we searched for laser systems emitting radiation where water is characterized by relatively high absorption. While other researchers have elected to pursue wavelengths of high absorption coefficient (8) and have recently reported alternative methods for de- livering these particular laser pulses to the zona target by either using glass micropipettes filled with positive air pressure (with the 193-nm radiation) (9) or using specialized fibers or micropipettes (in the case of the 2.94-txm erbium:YAG laser) (10), we have chosen to focus our attention on other wave- lengths. Possible difficulties with contact methods such as energy delivery to the target, the need for making use of a glass or salt-based instrument (some of them are toxic), and the necessity for maintaining physical contact between the delivery media (pipette or fiber) defeat most of the advan- tages in using laser light and essentially reduce the laser-based techniques to a mechanical/contact mode, similar to conventional PZD methods. Addi- tional complications arise from the need for steril- ization of optical fibers or micropipette tips. This requires constant adjustments in the delivery sys- tem and disposal of relatively expensive fiber seg- ments. The tip quality must also be carefully con- trolled in order to achieve a good beam quality, a consideration which is sure to slow down the pro- cedure. A final problem is due to the fact that some laboratories make extensive use of mineral oil cover over the targeted medium. As we discovered, drop- lets of oil tend to adhere to optical fibers when they are inserted through the oil layer into the host me- dium. These oil droplets, in turn, absorb the fiber output radiation, accumulate heat, expand (thereby mechanically disturbing the micromanipulated ob- ject), and completely block any additional radiation from arriving at its target. Based on the above considerations, we have elected to search for a set of laser parameters which will allow the laser light to be delivered to the Since their introduction, the techniques of zona drill- ing (ZD) and partial zona dissection (PZD) have drawn considerable attention as potential tools in gamete and embryo micromanipulation. ZD in- volves the application of small volumes of acidic Tyrode's medium, which, through its digestive properties, creates small holes in the zona pellu- cida (ZP). This process was originally described by Gordon and Talanski (1). PZD is a mechanical tech- nique for breaching the ZP and was first described by Malter and Cohen (2). It makes use of a pointed micropipette which pierces the ZP and is then sheared against a larger pipette, thus creating a breach in the zona pellucida. Indeed, most cur- rently accepted methods of micromaniputation of oocytes and embryos are based on the use of me- chanical techniques or the use of an acidified me- dium solution (3,4). In 1989, Tadir et al. (5) suggested the incorpora- tion of laser technology to achieve accurate inci- sions in the ZP as well as sperm micromanipulation through the use of optical traps. In addition, laser- induced selective destruction of extra pronuclei was also attempted (6). In these studies, the inter- action of the fundamental wavelength Q-switched Nd:YAG laser (15-nsec pulses of 1.064 ixm), the Nd:YAG harmonics (at 532 and 355 nm), and a nitrogen-pumped dye laser (600-psec pulses, at 366 nm) were all tested. In an effort to determine the most suitable param- eters for selective laser interaction with the various components within oocytes and embryos, we have been studying additional laser systems (7). Since cells in general, and oocytes and embryos in par- ticular, are relatively transparent and contain high The opinions presented in this column are those of its author(s) and do not necessarily reflect those of the journal and its editors, publisher, and advertisers, © 1992 Plenum Publishing Corporation