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15 Sep 2006
Researchers in the UK have found the optimum conditions for using a laser to create a hole that allows molecules such as DNA to be introduced into biological cells.
A femtosecond laser is an ideal tool for piercing a hole in a cell and allowing molecules such as DNA to enter, according to a team from St Andrews University, UK. Having tested the method on some 4000 Chinese hamster ovary (CHO) cells, the team believes it has come up with a set of conditions that offer the maximum chance of success (Optics Express 14 7125).
The art of using a laser to make a hole or pore in a cell membrane is called photoporation. Such a procedure creates an opportunity for DNA to enter the cell before the pore closes and the cell heals itself. If the DNA is then transcribed and translated into a protein, the process is known as optical transfection.
Biologists are becoming increasingly interested in photoporation, as this single-cell transfection approach offers a number of advantages over established techniques. Rather than transfecting entire populations of cells, optical transfection provides the ability to treat individual cells using a non-contact approach under sterile conditions.
"We have studied more cells than ever before to provide a detailed analysis of the optimum laser fluence required, the true transfection efficiency and the cellular viability of the technique," researcher Ben Agate told optics.org. "We have provided a realistic feel for the usefulness of femtosecond optical transfection in comparison to other techniques. Cells destroyed or irreversibly damaged were included in the data to provide a real and representative measure of the transfection efficiency."
Agate and colleague David Stevenson found that the transfection efficiency was strongly dependent on the laser fluence. At 1.2 µJ/cm², the average transfection efficiency was around 50%.
The duo used an inverted optical microscope equipped with a 1.5 W Ti:Sapphire laser operating at 800 nm and emitting 120 fs pulses at a repetition rate of 80 MHz. The laser's output was passed through a variable neutral density filter to control its intensity and a beam shutter to provide millisecond exposure times. Having expanded the beam to fill the microscope's objective lens, it was then focused to a spot of around 0.5 microns. A translation stage was then used to position each cell in turn.
Crucially, the cells were immersed in a solution containing plasmid DNA encoding for green fluorescent protein (GFP). Forty-eight hours after photoporation, fluorescence microscopy was used to calculate the transfection efficiency -- the number of cells successfully transfected and expressing GFP divided by the number of cells photoporated.
In their extensive six-month study, Agate and Stevenson varied the laser power from 50 mW to 225 mW and the shutter times from 10 ms to 250 ms. "A pore diameter of 1-2 microns, opening and closing within a few tens of milliseconds is optimal for optical transfection using femtosecond pulses at MHz repetition rates," said Agate.
The team has now received a Basic Technology Grant from Research Councils UK, which it will use to expand its research. "We have found that the efficiency of femtosecond optical transfection is around 50%, which is more than enough for most applications," said Agate. "This technique promises to be just as effective with human cells."
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