Laser trapping has been shown as a nondestructive way to manipulate individual cells, but what if the cell is still attached to living tissue? Creating enough laser power to disassociate the cell from the tissue sample can lead to overheating of the tissue and cellular death. A recent experiment highlighting the nonlinear effects caused by intense ultra-short laser pulses on biological material has led to the discovery of a new nondestructive optical method to isolate single cells from living tissue without damaging the cell from excess pressure or heat.
A team of researchers from Osaka University (Osaka, Japan), Kyoto University (Kyoto, Japan), and Japan Science and Technology Agency (Saitama, Japan) used shockwaves produced in culture media by nonlinear absorption of femtosecond laser pulses to disassociate single mouse cells from a prepared matrix.
The team's setup included a 20-Hz regeneratively amplified Ti:Sapphire laser with a center wavelength of 800 nm and a pulse duration of 120 fs connected to a microscope's optical port. "For our biological cells, we chose mouse NIH3T3 fibroblasts," says group spokesman Yoichiroh Hosokawa. "We plated them onto plastic coverslips coated with type-I collagen, and cultured them in a Dulbecco's modification of Eagle's medium (DMEM) containing 5% fetal bovine serum at 37°C." During the experiment, the chamber plate holding the cultured coverslips was also kept at 37°C, with a Peltier element and temperature controller. The team monitored cell appearance and locomotion via a CCD camera.
"Cells attach [to each other] with arms and legs called lamellipodia and filopodia," explains Hosokawa. "So to move a single cell, its 'podia must be severed. To do that, we gave each one a single laser shot of 0.26-µJ pulse energy." The shots caused the cell's filopodia to contract, but the cell did not exhibit the random motion expected of small particles suspended in gas or liquid (Brownian motion) -- in other words, the cell body was still attached.
Ultrafast laser pulses are used to disassociate a living cell from a matrix. During the first step, the cell's filopodia are cut by direct laser radiation (left); the cell retracts its filopodia (center); and the ultrafast laser pulse creates a shockwave that moves the cell a few microns from its original location (right).
The team then upped the laser pulse energy to 0.51 µJ, focused 20 µm from the ventral side of the cell, and fired. The laser shot's shockwave pushed the cell away, causing the cell to voluntarily detach from the matrix. The team was able to observe the Brownian motion of an isolated cell and determine that the cell was not damaged. A few hours after it detached, the cell regenerated its filopodia and once again adhered to the matrix. The team used a dye exclusion test to confirm the cell's viability.
Shockwave pressure is key to any damage to the living cell. Hosokawa says the force in nanonewtons of the shockwave, F0, can be estimated as a function of the pulse energy of the laser, I, in microjoules. The relevant equation, an experimentally determined law, is:
F0 = 6.99 X 103 X I2.17
The team assumes that F0 propagates uniformly in all directions from the laser focal point. That means the shockwave pressure, p, at the distance the cell is from the laser focal point, R0, is shown as:
p = F0/4πR20 = (6.99 X 103 X I2.17) / (4πR20)
Using this equation, when I = 0.51 µJ per pulse and R0 = 20 µm, the shockwave pressure on the cell should be 0.32 µN per µm2. "This force is much larger than that coming to bear on cells during conventional optical laser trapping," says Hosokawa. "However, we tried to move a cell with optical trapping, using 2 W of IR laser power, and it didn't work. What's more, the powerful laser irradiation raised the temperature of the culture medium, and could possibly cause critical damage. As our method causes no damage during cell detachment when the proper pressure pulses are generated at the fixed distance from the target cell, we feel it is definitely superior to laser trapping," says Hosokawa.
Hosokawa adds that a cell isolated by the team's nondestructive method can then be manipulated by conventional laser trapping, using a 400-mW IR laser. The research team feels that a combination of laser trapping and femtosecond laser-induced shockwave methodology will result in new possibilities in the single cell analysis of differentiation and tissue formation.
Saulius Juodkazis, researcher at the Research Institute for Electronic Science of Hokkaido University (Sapporo, Japan), says he is familiar with the work of Hosokawa's team. "I think that it really deserves attention. The method really works and holds excellent prospects in the field of cell research."