Laser Scissors and Tweezers T he intense, pure beams of light known as lasers are now stan- dard components of such com- monplace objects as compact-disc play- ers and printers. The everyday presence of lasers does not mean, however, that they have been reduced to performing only pedestrian tasks. Imagine focusing a beam specifically onto an organelle, a structure within a living cell. Consider further that the beam can actually grasp that minuscule entity and hold it in place. Now imagine that while this “micro- beam” acts as tweezers, a second beam serves as scalpel or scissors to conduct delicate surgery on the organelle. Even in a world accustomed to lasers, such musings have the ring of science fiction. Nevertheless, much as medical surgeons guide micromachined twee- zers and scissors through endoscopes to perform minimally invasive surgery on organs, the cell biologist can now use “laser tweezers” and “laser scissors” to perform minimally invasive manipula- tions on living cells and their organelles. Laser scissors came first. Almost three decades ago Donald E. Rounds and I, while at the Pasadena Foundation for Medical Research, suggested lasers might be wielded to probe the structure and function of cells and organelles [see “Cell Surgery by Laser,” by Michael W. Berns and Donald E. Rounds; Scien- tific American, February 1970]. Our early work focused on defining the pa- rameters of our lasers (such as wave- lengths of light and durations of expo- sure) and on determining which organ- elles could be successfully manipulated with light beams that could alter intra- cellular regions as small as 0.25 micron in diameter. (The diameter of an aver- age human hair is about 100 microns.) During the intervening years, my col- leagues and I found that laser scissors could be used to study organelles of the nucleus, such as chromosomes and the mitotic spindle that segregates chromo- somes during cell division. Lasers also facilitated studies of cytoplasmic con- stituents—namely, mitochondria (the energy factories of cells) and such struc- tures as microfilaments, microtubules and centrosomes—involved in maintain- ing cellular architecture and transport- ing molecules within cells. Putting Lasers to Work A lthough we do not always know ex- actly how lasers produce the spe- cific changes they make in cellular com- ponents, we can nonetheless generate certain alterations reproducibly and without compromising the target’s struc- ture or environment. For example, the traditional biological tools of light and electron microscopy show that laser scissors can produce a particular change in a chromosome, deep within a cell. Early work by our group demonstrated that scissors can inactivate a selected part of a chromosome in dividing cells— specifically, a region containing genes that control construction of a nuclear organelle known as a nucleolus. What is more, the alteration persisted in the cloned progeny of those cells, all of which possessed inactive versions of the genes in that same region. The alteration to the chromosome—a lesion less than a micron in size—ap- pears as a lightened region when the live cell is viewed under a phase-contrast light microscope. Careful transmission electron microscopy, capable of 10,000 to 100,000 magnification, reveals that same region to be a cleanly defined struc- tural alteration, with the chromosomal material on either side of the lesion, as well as the cytoplasm surrounding the chromosome, apparently unaffected. The “lightening” seen in light microsco- py is actually the result of a change in refractive index rather than a complete physical removal of material—the laser is changing the chemical and physical properties of the chromosome without totally destroying it. The cell membrane can likewise be studied, via a gentle perturbation of its fluidity. The membrane can even be in- cised, the laser cutting a micron-size hole that seals within a fraction of a second. Through this technique, called optopo- ration (pore production through optical means), molecules can be inserted into a cell when the pores are open without permanently damaging the membrane. Optoporation may be especially suit- able for genetic manipulation of plants, which have rigid cell walls that are rela- tively impenetrable compared with the supple membranes of animal cells. At the University of California at Irvine, my colleague Hong Liang and I have taken advantage of optoporation to insert genes into single rice cells; these geneti- cally modified cells gave rise to whole plants in which every cell carried and expressed the introduced genes. This work, when considered with the inacti- vation of nucleolus genes, demonstrates that laser scissors can be employed ei- ther to insert or to delete genes. In Europe, laser-scissors manipulation of gametes (sperm and egg) has been ap- plied recently in the human clinic, as part of a procedure called assisted hatching. The scissors thin or remove a small area of the protective zona pellucida of eggs that have been fertilized in a laboratory dish. The very early embryos are then placed in the womb, where the thinning of the zona appears to abet implanta- tion. Thinning can also be accomplished by more conventional techniques, but the laser method works without toxic chemicals that can damage the embryo. The most extensive human study to 62 Scientific American April 1998 Laser Scissors and Tweezers Researchers are using lasers to grasp single cells and tinier components in vises of light while delicately altering the held structures. These lasers offer new ways to investigate and manipulate cells by Michael W. Berns Copyright 1998 Scientific American, Inc.