Green Fluorescent Protein (GFP) does exactly what its name implies: when excited by light from the blue end of the spectrum, it emits a satisfying green glow. But its deceptively simple name doesn't begin to convey how it has changed biology. It can be fused with other proteins and made by just about any cell, letting researchers track all sorts of biological processes. Variants have been made that glow other colors. Three people got Nobel Prizes for its development. Glowing animals have even become works of art and pets.
And now it has turned a cell into a laser, although researchers had to put mirrors on either side of the cell to get it to work.
Making something into a laser requires the creation of what's called a population inversion. For most systems, the majority of the molecules present sit in the ground state; only a few are excited to higher energies. To get a laser to work, however, it has to be possible to invert that, and place most molecules in the excited state. That way, when a photon of the appropriate wavelength hits a molecule, it will be in the excited state and not be able to absorb the photon; instead, the excited molecule will emit a photon and drop to the ground state.
GFP, the authors of the new work reasoned, has some properties that make it a good candidate for the sort of gain material that makes a population inversion possible. It can absorb a broad range of wavelengths to put it into an excited state, after which it quickly drops to the lowest energy excited state without emitting any photons; it will stay in this state for up to a few nanoseconds. From there, it can undergo stimulated emission of a photon, which it does with something close to an 80 percent quantum efficiency. Provided a pump can keep things cycling to the excited state faster than that handful of nanoseconds (and the authors have access to a laser that can), GFP should be able to lase.
They started off conservatively, working with a solution of purified GFP protein. (They don't list their purification procedure, but it presumably involved keeping any solutions that glowed green.) A drop of the resulting GFP solution was then placed on a mirror with a hydrophobic surface. A second mirror was brought into contact, and then pulled back until it was a few millimeters away; the surface tension kept the drop suspended between the two mirrors. They then began hitting the drop of GFP solution with a pump laser to keep the protein population in an excited state.
Once the pump laser's energy went above 14 nanoJoules, the output energy "rose dramatically faster with increasing pump energy," and the device emitted a green light that was visible to the naked eye. Instead of the relatively broad emission of the native protein (which can release photons from a variety of excited states), the GFP laser's emission was quite narrow, indicating that most of the photons were being emitted from the lowest-energy excited state. The authors found that they could get lasing with protein concentrations as low as 2.5 microMolar.
And that must have gotten them pretty excited, since the GFP concentration inside cells get into the milliMolar range, quite a bit higher than needed for lasing. So, they took a kidney cell line and inserted DNA that encoded GFP into the cells. They estimate that these cells had internal GFP at concentrations of nearly 300µM, well above the requirements for purified protein. A suspension of the cells was then placed next to two narrowly separated mirrors—capillary action sucked the cells (and some of their growth medium) into the space between the mirrors.
A microscope was then used to locate and stimulate individual cells, which proceeded to lase. This required less than a single nanoJoule of stimulation energy, lower than the requirement for the protein solution. The energy was low enough that the cells survived the whole procedure—when they were done lasing, they could be put back into culture and grown further.
Instead of a single point of emission, however, the cells showed a number of distinct internal areas with intense emissions, and these were often at distinct (though similar) wavelengths. Using a diffraction grating, the authors were able to separate out the individual modes of the cellular laser, and found that different cells created distinct patterns. "The exact patterns and eccentricity of the modes result from the specific cell shape and the gain and refractive index profiles within the cell," the authors conclude. In other words, the light that comes out of the cell provide some information about what the cell is structured like internally, although the authors didn't look into how much you could infer about the cell's structure from this output.
It's an amazing piece of work, and the only thing that spoils it a bit is the discussion. The lasing does provide some information on the structure of the cell, but it's not clear that we can't get even better information from simply imaging the GFP directly, since the protein won't end up evenly distributed throughout the cell. Even more speculatively, they suggest that this technique could be adapted to work inside living organisms without the need for mirrors. This bit of speculation would require getting lasing with surface plasmons to work, which we already know faces a lot of its own issues.
We'll have to wait for the authors (or someone else) to determine if the laser's profile can actually be used to extract structural details that elude more conventional imaging techniques. In the mean time, we can console ourselves with the thought that they've done something phenomenally cool.
No comments:
Post a Comment