This year’s Nobel Prize in Chemistry, announced just a couple of weeks ago, honored three scientists — Osamu Shimomura, Marine Biological Laboratory (MBL), Woods Hole and Boston University Medical School, Martin Chalfie, (Columbia University), and Roger Y. Tsien, University of California, San Diego, “for the discovery and development of the green fluorescent protein, GFP, ” most notably seminal work to design and create fluorescent molecules that enter cells and light up their inner workings.
GFP was first observed in jellyfish in 1962. Since then, it has has become one of the most important tools used in contemporary bioscience. With the aid of GFP, researchers have developed ways to watch processes that were previously invisible, such as the development of nerve cells in the brain or how cancer cells spread.
Genetic tweaking is one application. The pretty green fluorescent protein made a bit of a splash last year when Korean scientists at Gyeongsang National University in Jinju cloned a Turkish Angola kitten, modifying the three resulting copies genetically with GFP to change their skin color. When viewed under ultraviolet light, the cloned kitten gave off a red fluorescent glow, while the original kitty appeared to be green.
But by far the more critical application of GFP has been its use as a tagging tool in bioscience. By using DNA technology, researchers can now connect GFP to other interesting, but otherwise invisible, proteins. This glowing marker allows them to watch the movements, positions and interactions of the tagged proteins, without disrupting or harming the cell, opening new windows into cellular function. Previously, dyes were injected, violating the cell membrane and limiting studies to larger cells.
Fluorescence microscopy is pretty much the standard workhorse for biological imaging these days, but it has limitations, most notably in its ability to resolve and focus on featres smaller than optical wavelengths. Ergo, there are lots of research groups developing ever-better versions of the basic instrument to overcome these limitations. Harald Hess of the Howard Hughes Medical Institute was on hand for this morning’s IPF session on bio-imaging to talk about his new bio-imaging technique, Photo-Activated Localization Microscopy (PALM).
Bio-imaging has two main issues. First is the need for sub-optical resolution mentioned above. Second, it requires a biocompatible means of achieving specificity — that is, there must be a way to enhance the contrast between different proteins, which otherwise pretty much all look alike. GFP provides that contrast, along with other, subsequently developed photo-activatible fluorescent proteins — it is the “magic bullet” that makes PALM possible, says Hess.
With PALM, successive sparse subsets of fluorescent proteins like GFP can be activated individually, then excited to cause them to fluoresce so they can then be imaged. And this process can be done repeatedly, accumulating the data to resolve it into a highly detailed PALM image. He’s demonstrated the technique on a nice sampling of biological molecules thus far, including mitochrondria, lyosomes, actin networks, and other cell structures.
The next step for Hess and his colleagues is the development of dual color PALM imaging, as well as a related technique combining PALM with an interferometric microscope to measure the vertical position of fluorescent molecules. Dubbed iPALM, this technique can provide fully three-dimensional imaging of proteins with resolutions as small as 20 nanometers. Hess would also like to increase the imaging speed of PALM-based techniques to achieve live cell imaging and motion tracking of molecules, with the aim of directly observing how molecular transport mechanisms work in situ.
Maybe it’s not as mainstream media-friendly as glowing cloned kitties, but PALM nonetheless makes some awfully pretty pictures.
