“Lead, kindly Light, amid th’encircling gloom;
Lead thou me on!
The night is dark, and I am far from home;
Lead thou me on!
Keep thou my feet; I do not ask to see
The distant scene–one step enough for me”
Lead, Kindly Light
– John Henry Newman
Edited by Navjot Kaur
Imagine this – you’re a researcher (sounds easy, eh?) looking desperately for a protein within a cell. Your job is to track it, see how it moves around within the cell, observe its interactions, or more importantly, figure out whether the cell you’re looking at even has it!
Quaintly niche as this could sound, this does happen to be an eternally recurring question in biology. Over the decades, pioneering techniques of awe-inspiring creativity have been designed to help tackle this very issue. Even today, scientists are ever on the lookout for improvements or innovations which could further refine the scales at which we can currently see, constantly pushing the boundaries of the depths to which technology can penetrate.
Fluorescence-based detection methods stand out as a milestone in this quest of exploration. Though its current spectrum of applications has far outstripped its original raison d’etre, it stands as a testament to how basic concepts of physics can be harnessed to provide powerful tools for the wily biologist.
Almost everyone is familiar with the general idea of fluorescence (NOT to be confused with phosphorescence). Well, maybe its worthwhile to make the distinction while we are at it.
Phosphorescence is what you see on your glow-in-the-dark watches and shirts. A material which is phosphorescent would absorb light falling on it to a great extent, and gradually keep on emitting this energy once the source of light has been removed. Essentially, a fluorescent object would also do something similar. The key difference being that the phosphorescent object tends to absorb light energy to a much greater extent, and loses it way more slowly than a fluorescent one. The “glow” we observe as a result of this, may hence last for extended periods in case of phosphorescent objects – the reason why your watch dial will glow all night-round if you’ve exposed it to light during the day. Whereas, fluorescent objects usually stop emitting light almost as soon as the source exciting them in the first place is removed.
Now think about this analytically – wouldn’t a fluorescent particle be a lovely way to override the difficulties mentioned in the opening para of this piece? If you could, by some conceivable means, stick a fluorescent particle onto your protein of interest, you could observe it moving around every time you excite it with a light source. And voila! problem solved. An invisible protein, too small to visually see under the microscope, is now being heralded by a tell-tale light beam tagging along with it.
Such a particle would be akin to a GPS tracker on your mobile. As long as it’s on, it’s easy to track your movements. You could also turn it on and off at will, as the particle emits light only as long as you provide it an exciting source in the first place.
Reality, as always, happens to be a lot more complicated!
For the longest time, getting a fluorescent particle into a cell was a formidable challenge. You couldn’t just pick up a tiny fluorescent bead and stick it into a cell – such a foreign body would never be accepted as a part of the cell. Scientists needed to figure out a way to make the cell treat a fluorescent particle the same way it treated its own components – or else, any behavior you observe might well just be an isolated effect towards a foreign body.
Although fluorescent dyes and stains have been around for long, the real breakthrough with respect to “tagging” cellular components like proteins with a fluorescent entity came about in the 1960s. It was a young Osamu Shimomura, fresh from a PhD at Nagoya University and working as a researcher at Princeton, who successfully managed to isolate the G.F.P. (Green Fluorescent Protein) from jellyfishes. It was certainly no cakewalk – he had to sift through thousands of other cellular extracts before finally getting his hands on a miniscule amount of the protein which glowed green when light was shone on it.
An intense period of optimization, application, and refining followed as over the next few decades, not only did we literally have an explosion of colors (Yellow, Red, Cyan, Orange, so on), as well as breathtaking strides in their spectrum of applications. Let alone proteins, scientists would go on to create even the most seemingly outlandish of fluorescent entities – glowing pigs, sheep, rabbits …. The sheer creativity of innovation can appear almost ludicrous.
Coming back to the question we asked – how would we track our elusive inhabitants within the cell? Shimomura’s discovery of a naturally occurring fluorescent protein was but the first step. Jellyfishes aren’t humans, and certainly scientists were not going to shift over from a century of using cells and rats as models to jellyfishes overnight. And that’s when the ever busy minds of molecular biologists and biotechnologists came together.
Shimomura had played the part of Columbus – the New World was here (yes, I am aware of Vespucci, but hey, let’s keep it this way). The explorers and settlers would now follow. Ironically enough, Shimomura himself wasn’t much interested in using the GFP as a tracer molecule.
In stepped Douglas Prasher, who reasoned that if regular proteins could be made to carry a GFP element as a tag which would then be a part of the protein itself, it would be a simple step up to use it as a tracer molecule – wherever the protein went, the GFP would follow by tagging along. Using Shimomura’s GFP as a reference, scientists first tracked down the gene responsible for producing this protein. Once this was found, it was only a matter of clever manipulations before they managed to insert this gene into the DNA of other cells – bacterial, insect, mammalian – and making these cells express the GFP independently.
It was a startlingly intuitive, yet utterly remarkable piece of innovation which pieced together two seemingly disparate fragments of knowledge to birth a powerful new tool.
This realization was a key breakthrough – GFP could now be produced within cells, and hence would be treated as protein of the cell itself. Fast forward a bit, and they were now achieving what Prasher envisioned – inserting the gene for GFP as a part of the gene for a specific protein itself, so that whenever this protein was made, the GFP component would be automatically made as well as an extended part of the original protein itself.
Despite the revolutionary contribution, Prasher’s story ended up in a tragic heartbreak. Though his initial work in these directions, in collaboration with Martin Chalfie and Roger Tsien, took off well enough, a lack of funding forced him further away from the project, and led to his untimely exit from academia. Though Prasher demonstrated incredible fortitude in the face of adversity – even carrying on the research unfunded for a short period – it ultimately became untenable. Eventually, he had to find work as a shuttle bus driver – a far cry from the astronomically successful career path that might have otherwise lain ahead. In a bittersweet conclusion, Shimomura, Chalfie, and Tsien were awarded the 2008 Nobel Prize in Chemistry for the very work which Prasher had been such an integral part of. Subsequent recognition notwithstanding, it does remain as a singularly cruel twist of fate for a man who had ushered in one of the most significant modernizations in biology.
The ramifications of such an innovation were, and continue to be, unbelievably far-reaching. We can now latch similar fluorescent entities onto everything ranging from individual proteins, cell membranes, nuclear components, sub-cellular organelles, and so on. Researchers have exploited the tracking abilities of GFP and similar proteins to painstakingly track proteins all across cells, understand the intricate pathways that go into manufacturing, packaging, and polishing a protein, study diseases, comprehend the behavior of sub-cellular entities, and in short, investigate almost every conceivable aspect of cell biology.
Today, we’ve come a long way from Shimomura and Prasher’s original GFP. Synthetic analogs are now being widely used, as well as modified, highly improved substitutes which have remarkably extended the boundaries of what a simple tracer molecule is capable off.
Even in my own project, we use GFP-tagged cancer cells on a routine basis, as do many of the other members in the lab. They’re surprisingly convenient to image since they show up prominently under appropriate lighting conditions – thereby making it all the easier for us to analyze their every aspect. It’s a ridiculously straightforward method to focus your attention and almost innocuously shut out the rest of the world.
So often it becomes a habit to let familiarity breed contempt, and to take the toils of the pioneers for granted. At our lab, we grow these tagged cells and culture them by the thousands, almost on a daily basis, but rarely ever even consider what an incredible feat of ingenuity it took to engineer the now staple workhorse.
But sometimes, just that one rare moment arises, when for a second distracted from the constant worry about exposure times and camera settings, you just gaze down the eyepiece and see the shimmering green delights below – marveling in their muted elegance and dumb beauty – and recollecting your instructor’s tale of how an unsuspecting jellyfish turned into the kindly light that led science towards some of its greatest triumphs.
Sleepy Cats and Falling Apples 