Imagine latching onto a wiggling sperm cell, holding it in place and then gently guiding it through a tiny opening into an egg cell (oocyte). Using two lasers and an inverted microscope, biophysicist Dr. Karin Schütze, head of the Applicative Laser Unit at Klinikum Harlaching, a hospital near Munich, Germany, has done just that.
Optical tweezers and more
The system, developed with Raimund Schütze at the Institute of Scientific Film in Göttingen, teams a laser microbeam to penetrate the protective layer around the oocyte and a laser microbeam that acts as "optical tweezers" to hold and deliver sperm, a far less invasive approach than traditional micromanipulation. It is called positioning and ablation with the laser microscope (PALM).
"The two different lasers are coupled through the same port into the microscope. A double-beam optical trap was developed using two different trapping beams. All laser beams can be moved in the x, y and z direction, independent from each other, the microscope or the microscope stage," explains Dr. Schütze.
Schütze has been working with noncontact optical micromanipulation for about 12 years, having trained with Art Ashkin at the University of California, Berkeley, to learn optical trapping. "When I returned to Germany, I combined the technique of highly precise laser cutting with optical tweezers and the application of optical micromanipulation was extended to in vitro fertilization procedures," she says. PALM uses different wavelengths from the Ashkin technique.
It started with Louise
To appreciate the significance of Schützeís work, it helps to look back to July 25, 1978. A very special little girl, Louise Joy Brown, was born that day at 15 minutes before midnight. The story actually began 39 weeks previously, when Louise was conceived in a laboratory dish. She has led a pretty normal life, considering that she was the worldís first "test tube baby," born via in vitro fertilization (IVF).
While IVF has brought children to thousands of couples suffering from infertility, it has proved disappointing to many more, with a success rate at most clinics of only about 14 percent. The several tries it usually takes to become pregnant add up to thousands of dollars.
Two other techniques improve the odds. For gamete intrafallopian transfer (GIFT), sperm and oocytes are placed in the womanís fallopian tube, allowing fertilization in her body. The success rate is 26 percent, and the cost is half that of IVF. For zygote intrafallopian transfer (ZIFT), a fertilized ovum is introduced into the fallopian tube, with a 23 percent success rate.
GIFT and ZIFT arrived in the mid-1980s. The 1990s have seen a variation on the assisted reproductive themeñhelping sperm. This entails either drilling a hole in the zona pellucida, the layer of proteins and carbohydrates that shields the oocyte ("zona drilling"), or injecting a sperm cell through the zona ("intracytoplasmic sperm injection" or ICSI). These two techniques bring the possibility of parenthood to men whose sperm lack tails or cannot move for other reasons, and to men who cannot ejaculate because of spinal cord injuries.
Enter PALM
PALM enters the picture against this backdrop of increasing success in assisted reproductive technologies. Because the laser microbeams do not cut through cells and do not introduce pipettes and other intrusive devices, they have the potential to improve the effectiveness of zona drilling.
Previous work on mice had shown that using a 308-nm excimer laser to drill a hole in the zona increased the fertilization rate sixfold, but the fertilized ova often did not develop further, perhaps because this wavelength damages DNA. Instead, Schütze and her co-workers used a 337-nm pulsed nitrogen ultraviolet laser microbeam to make the hole. Pulse duration was 3 ns, for 15 pulses per second. This created a hole 5 to 15 µm in diameter in the zona.
The optical tweezers that captured sperm came from a diode-pumped Nd:YAG laser of 1064-nm wavelength. Mice were apparently unharmed by the double-laser procedure, giving birth to normal pups that, in turn, grew up and had normal pups. An added touch is to use the ultraviolet laser to zap the tails off the sperm, making them easier to catch.
In a paper published in the March 1996 Journal of Assisted Reproduction and Genetics, Schütze and her colleagues report using the technique on cow oocytes obtained from slaughterhouses. The researchers brought together sperm and oocytes in a variety of circumstances, counting as a successful fertilization evidence of two nuclei and a sperm tail within the oocyte. The first group of cells was oocytes plus the "cumulus" of cells surrounding them. These were exposed to densely packed sperm (a million per milliliter of ejaculate), yielding a 53.9 percent fertilization rate. The second group, oocytes minus cumulus in the presence of dense sperm, yielded 50 percent fertilization. The third group consisted of oocytes, one hole laser-drilled in each zona, and dilute sperm (5000 cells per milliliter), yielding zero fertilization. The fourth group had oocytes with zona holes plus three to five sperm that were immobilized and inserted using optical tweezers. Here fertilization was 3.8 percent.
Group 3 served as a control for Group 4, demonstrating that it was the manipulation of the sperm that accounted for fertilization, because sperm left to swim about on their own near a zona hole didnít get the job done. "We could prove that fertilization was only due to the laser-introduced sperm," Schütze says.
The PALM Combi-System, which includes the optical tweezer and microbeam setup, can also examine cells of eight-celled pre-embryos for genetic defects, activate pre-embryos by piercing their coverings and cut chromosomes. But for now, Schütze is focused on zona drilling as a way to improve the efficiency of IVF. "In humans," she says, "fertilization rates could be significantly increased in a large group of patients after laser zona drilling ... and subsequent mechanical sperm injection by use of [a] laser."
For further information, contact; Institut für den Wissenschaftlichen Film, Nonnenstieg 72, D-37075 Göttingen, +49 55 1 50240; FAX, +49 55 1 5024-400.
By Dr. Ricki Lewis Contributing Editor
feedback form |
permissions |
international |
locate your campus rep |
request a review copy
Copyright ©2001 The McGraw-Hill Companies.
digital solutions |
publish with us |
customer service |
mhhe home
Any use is subject to the
Terms of Use and Privacy Policy.
McGraw-Hill Higher Education is one of the many fine businesses of the
The McGraw-Hill Companies.