Epifluorescence microscope image of mitochondria before and after laser disruption. By using an impermeable dye, ethidium bromide, that can only enter a cell with a damaged cell membrane, researchers can eliminate internal subcellular structures without harming the overall cell. (Harvard University)
Organelles and other subcellular structures are the machinery by which cells carry out their basic functions. Cellular biologists typically use biochemical and genetic techniques to change one structural protein at a time when investigating subcellular structures and their effect on the cell's operation. However, because of the complexity of the biochemical and genetic processes in a cell, it is difficult to isolate subsequent effects caused by the chemical or genetic change with a high degree of certainty.
Photonic techniques such as fluorescence microscopy and laser tweezers have allowed cellular biologists to manipulate cells in the past but not to remove subcellular structures while leaving the rest of the cell intact. All that has changed, however. Eric Mazur and colleagues at Harvard University (Cambridge, MA) and Harvard Medical School have used tightly focused femtosecond laser pulses to vaporize individual organelles and other subcellular structures with submicrometer precisionwithout disturbing the rest of the cell.
"Although they've been around since the '80s, it's not until the last five years that femtosecond lasers have been simple enough to operate to encourage this kind of cross-disciplinary work," says Chris Schaffer of the University of California at San Diego's biochemistry department and organizer of the Commercial and Biomedical Applications of Ultrafast Lasers IV Conference at Photonics West 2002 (1925 January; San Jose, CA), where Mazur's group will present its latest work (paper #4633-29). "Sometimes you want to genetically or immunicalogically affect all of one type of substructure, but other times you just want to affect a few substructures of one type within a cell and see how the rest react. This is the only way to accomplish that."
Nan Shen, lead researcher for Mazur's group, uses a continuous-wave (CW) 5-W diode-pumped solid-state laser (Coherent Inc.; Santa Clara, CA) to pump a titanium-doped sapphire (Ti:sapphire) oscillator (Kapteyn-Murnane Laboratories L.L.C.; Boulder, CO). The setup also includes a neodymium-doped yttrium lithium fluoride (Nd:YLF) second-stage amplifier that produces a 1-kHz pulse train, but Shen intends to remove the amplifier from the setup as soon as possible and use software to control the repetition rate of the Ti:sapphire. "It's only there because we share the setup with other experiments," Shen says. "For our purposes, we only need 2 nJ per pulse, so a second-stage amplifier isn't necessary." An epifluorescent microscope with high numerical aperture (NA 1.4), oil-immersed objective delivers the pulses to the cell.
The trick, according to Shen, is to deliver just the right amount of energy: too much and you disrupt the entire cell, too little and the 800-nm light from the Ti:sapphire passes right through the sample. Each 100-fs pulse is focused down to an hour-glass shaped area some 400 nm in diameter at the waist and 1 µm deep. The high NA lens creates an 80° cone angle for each laser pulse, compressing the bulk of the laser light to a focal point smaller than the wavelength of the laser light. The sheer number of photons at the focal point encourages nonlinear multi-photon absorption of the photons by the electrons in the subcellular structure. After a few pulses, enough light is absorbed to either vaporize or disrupt the structure. Shen can then view the results on the epifluorescent microscope or take the sample to a nearby lab for confocal imaging.
Early results are encouraging. With greater control of the pulse repetition rate, Shen intends to move from epifluorescent imaging to a high-resolution real-time multi-photon microscope, using some of the pulses for disruption and the rest to excite longer-lived fluorescent dyes that will allow her to watch the disruption in real time.
"There are confocal microscopes that operate at full video rate," Schaffer adds. "All the pieces are there to watch cell repair mechanisms and intracellular signal transduction processes in real time."