14 Apr 2009
The next time you look at a list of microscope accessories, a photoporation module for cellular analysis could well be available. Jacqueline Hewett talks to Kishan Dholakia about the clinical and commercial potential of optical injection by photoporation.
Photoporation is an interdisciplinary technique that is oozing with potential. Not only could it unlock secrets in areas ranging from pharmaceuticals through to agriculture, but the simplicity of the method means that it could be turned into a practical accessory that is suitable for use with modern confocal microscopes.
"I think photoporation will become a ubiquitous technique in the next five years," Kishan Dholakia, who is developing the technique with Frank Gunn-Moore at the University of St Andrews, UK, told OLE. "The time for this field is now. The lasers are getting better, more reliable and less expensive. We now need more groups to take it onboard and do more rigorous studies of different cell types. Some amazing biophysics is going to be opened up with this technique."
What is photoporation?
Photoporation is a way of delivering foreign material such as drugs or DNA into a cell in a targeted and sterile manner. Put simply, photoporation is the process of punching a micron-sized hole in the cell membrane. If a membrane-impermeable substance enters the cell during the time the hole is open, then optical injection has occurred. If the membrane-impermeable substance is a nucleic acid species (such as DNA or RNA) and the cell subsequently expresses the protein encoded by the species, then optical transfection has occurred. The cell then heals itself and scientists wait to see the results.
Although there are other transfection methods, the real benefit of the optical approach is its ability to target single cells. "If you are a pharmaceutical company, you wouldn't have to develop a large volume of a drug and test it on lots of tissue," commented Dholakia. "You can start at the cellular level and it really reduces the cost."
Because the vast majority of this work is already done under a microscope, it lends itself to an optical solution such as optical injection. "The beauty is that this could be an add-on to a conventional microscope," commented Dholakia. "In future, we hope to have an optical injection module that could be fitted onto any microscope. Anyone could image cells and transfect them."
The optical syringe
Over the past five years, the St Andrews team has worked on various laser systems to find the optimal way of piercing the cell membrane. Today, its method of choice is a modelocked Ti:sapphire laser emitting 100 fs pulses at a repetition rate of 80 MHz – a common piece of kit.
"We irradiate the cells for tens of milliseconds at an average power of tens of milliwatts," said Dholakia. "The light is focused close to the cell membrane using a standard microscope objective. This creates free electrons near the cell membrane that photochemically react and cause a hole to appear."
One of the major hurdles has been the need to maintain the beam focus precisely on the cell membrane. Techniques such as optical injection are multiphoton processes. This means that, due to a nonlinear intensity dependence, the process only occurs over a very short axial range, which gives rise to stringent focusing requirements. Locating the exact position of the cell membrane is a time consuming and undesirable task, one that the St Andrews team was keen to remove.
The solution was an in-depth study of alternative approaches that use non-diffracting elongated beams called Bessel beams. Such beams comprise a central maximum surrounded by a set of concentric rings with increasing radius. Crucially, the central maximum of a Bessel beam does not diffract over a long range. So, by using a Bessel beam, a relatively small spot size can be maintained over a relatively long beam path. This significantly reduces the need to focus the beam to an exact position on the cell membrane.
"We call this method an optical syringe," said Dholakia. "A Bessel beam is a good way to create a rod of light so that you don't have to focus the beam very tightly. It's also a practical solution for non-optics professionals who don't want the hassle of working with optics and focusing the beam."
Adding to the practical nature of the technique, the group has also developed user-friendly point-and-click software that essentially runs the experiment at the click of a button. "The software talks to a spatial light modulator," explained Dholakia. "You click on the cell that you want to transfect and the laser is directed by the modulator to the cell and punctures it."
With all of this technology in place, the big question is: does it work? Do the cells survive the transfection process?
"We typically get an efficiency of 40–70%, which is very good," commented Dholakia. "This efficiency is an indication of cell viability. This is where we would optically inject an individual cell, inserting a gene encoding for green fluorescent protein. The cell takes it up, fluoresces green and doesn't die afterwards."
Optimization and applications
With all of the initial groundwork complete, knowing where to go next is a tough choice as there are so many avenues of investigation open to Dholakia and his colleagues. Ongoing work now is a mixture of further laser studies and researching various cell lines and applications in biology.
"It's good to keep pushing to optimize the laser parameters and understand the mechanisms better," commented Dholakia. "For example, we are also investigating optical injection with 405 nm laser diodes running continuous wave. This works, but we believe that it is due to more of a direct heating effect. Whether it is better or worse is still in question, the jury is still out but it is still a very exciting area." In a similar vein, Dholakia believes that there also needs to be comprehensive studies looking at the benefits of using picosecond and nanosecond lasers. Certain lasers might be better for certain tasks within optical injection, for example.
One future development could be fibre delivery and the introduction of dispersion compensation. "We have also developed a fibre-based optical injection prototype that is compatible with a standard endoscope," said Dholakia. "Maintaining a short pulse duration using dispersion compensation would reduce the average power needed. It adds a level of sophistication to a system that is already very practical and uses standard laser systems."
In terms of biological applications, Dholakia collaborates with St Andrews-based neuroscientist Frank Gunn-Moore. "We are looking at cell-to-cell signalling and aspects within the development of Alzheimer's disease," explained Dholakia. "We are working on brain slices, which is exciting as the cells involved are not very easy to isolate using other methods. Optical injection is the ideal way to study diseases. A lot of cell types are difficult to transfect, and that's where people should be thinking about this technique. All you have to do is tailor the laser parameters to the cell line that you want to study."
• This article originally appeared in the April 2009 issue of Optics & Laser Europe magazine.
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