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Tag, you’re it! A new process provides a better understanding of cells

Scientists hook fluorescent tags onto molecules to see how they keep cells alive. Such tags used to burn out quickly. Now they glow longer, revealing more about how life works.

Video animation of fluorescent tags on cells

Long-lasting fluorescent protein tags can track molecules in cells for an hour rather than seconds.| Video courtesy of Rajarshi Ghosh, Drea Sullivan 

Anyone who has watched a time-lapse movie knows how it can condense an achingly slow process into mere seconds.

Previously unknown patterns emerge. Hidden relationships become obvious.

Now, a team led by Stanford bioengineer Jan Liphardt has developed a way to make time-lapse movies of the chemistry of life by following individual molecules as they move through cells over an extended period. They did this by improving on a relatively new technique that allows scientists to tag biological molecules with fluorescent proteins to track their motion. If we think of the cell as a machine, and molecules as its moving parts, it is only by seeing where molecules go and what they do that we can understand how life works. By improving these fluorescent tags, the Stanford researchers have found a way for scientists and engineers to have a better understanding of biological processes.

Fluorescent protein (FP) tagging has been so useful that its discoverers shared the 2008 Nobel Prize in chemistry. The technique enables biochemists to attach FPs, or fluorophores, to a molecule that they would like to follow. When illuminated by laser light, the fluorophores glow, allowing researchers to track the tagged molecules. But the current technique has had a critical shortcoming. Fluorophores typically burn out within seconds through a process known as “bleaching.” Thus, tagging has, so far, only given researchers tantalizing glimpses of molecules in motion. Liphardt likens this to trying to study traffic flows on a highway at night when headlights only stay on for a few seconds.

To beat this time limit, Liphardt and Rajarshi Ghosh, a postdoc in his lab, developed a new type of fluorescent tag that, for all practical purposes, never burns out. They base their technique on two innovations that work together, as the authors explain in the journal Nature Chemical Biology.

First, they attach nanoscale arrays to the biomolecules they want to study. These arrays act like scaffolds that many copies of fluorescent proteins, or fluorophores, can glom onto. Second, and counterintuitively, they used weaker fluorophores that become brighter as they attach to the arrays on the target molecule. Once bound to a nanoscale array these dimmer fluorophores collectively become up to 600 times brighter than the background.

The Stanford fluorophores achieve their relative brightness in two basic ways. First, they accumulate many weaker lights to create a strong glow. Just as importantly, unattached tags floating around in the cell emit less distracting light. The starker contrast between tagged molecules and the cellular background allows researchers to use weaker lasers to illuminate the fluorophores. Weaker lasers, in turn, bleach the fluorophores more slowly, extending their useful lifespan. Best of all, when one of the dim fluorophores does bleach out, it drops off the array and an unattached fluorophore takes its place, maintaining the overall brightness.

To establish the endurance of their process, the researchers tracked single tagged molecules for an hour and captured microscopic images at high frame rates to make a time-lapse movie. They say the only practical time limit on the process is how long the cell will tolerate being probed by the laser. The technique enables scientists to go from producing seconds-long snippets to feature films of molecular interactions.

Recalling a quote by the physicist Richard Feynman, Ghosh says that the key to understanding nature is continuous observation, like learning chess by observing masters at play. The rules may be a mystery at first, but we come to know by continuing to watch.

“Using this technique, we can see events unfold in a molecule’s life as a continuous thread, instead of trying to reconstruct complex pathways from glimpses,” Ghosh says.

Liphardt says the team would next like to make the technique even more useful by developing a whole family of fluorophores that can work in different light spectrums. This would enable researchers to study multiple reactions simultaneously, using fluorophores that glow in different colors to reveal the complex relationships between various biological molecules.

In the meantime, Liphardt and Ghosh are finding it hard to keep up with demand from the research community eager to use their new fluorophore arrays to make movies of molecular life.

“We can’t ship things out fast enough,” Liphardt says.