ABSTRACT
Practical advantages of using femtosecond laser pulses for manipulations in cell surgery were demonstrated. The use of femtosecond laser pulses enables precision punching of the zona pellucida of the embryo without damaging its cells. With the help of femtosecond laser tweezers/scalpel, auxillary laser hatching was performed and a technique of optical biopsy of mammalian embryo was developed, which enabled non-contact sampling of embryonic material for preimplantation diagnostics. Our findings suggest that about 90% embryos retained the ability to develop at least to the blastula stage after this manipulation.
Subject(s)
Lasers , Microsurgery/methods , Optical Tweezers/adverse effects , Preimplantation Diagnosis/methods , Animals , Biopsy , Embryo, Mammalian , Female , Mice , Microscopy , Microsurgery/instrumentation , Pregnancy , Preimplantation Diagnosis/instrumentation , Zona Pellucida/ultrastructureABSTRACT
Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (Manson et al.,1980. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.
