Posted in | Chemistry

New Revolution in the Field of Single-Molecule Imaging

Chemists do not have to chase rainbows anymore as a new technique to craft a spectrum of glowing dyes has been discovered.

These are novel rhodamine dyes synthesized in the Lavis Lab fluorescing under UV illumination Credit: Jonathan B. Grimm

In the journal Nature Methods, Researchers report that practically any color Scientists’ desire can be produced - ROYGBIV and beyond, when certain chemical building blocks in fluorescent molecules known as rhodamines are swapped out.

The study provides Researchers a way to consciously adjust the properties of existing dyes, making them brighter, bolder and further cell-permeable also. Study leader Luke Lavis, a group leader at the Howard Hughes Medical Institute's Janelia Research Campus in Ashburn, Virginia, says such an expanded palette of dyes could help Scientists to illuminate the inner workings of cells even better. His team made larval fruit fly brains shine, lit up cell nuclei, and emphasized visual cortex neurons in mice that had small glass windows integrated into their skulls.

Researchers used to concoct different dyes largely by trial and error, Lavis says. "Now, we've figured out the rules, and we can make almost any color." His team's method will possibly enable Chemists to synthesize hundreds of different colors.

A bright history

Scientists depended on chemical fluorescent dyes to make biological molecules visible about two decades ago. In July 13th, 2017 perspective in the journal Biochemistry, Lavis wrote that, “chemistry was king” for peeking inside cells, staining organelles and other imaging experiments. The king was then kicked off the throne - by a glowing green jellyfish protein known as GFP.

Researchers in 1994, reported the use of a genetic trick to tack GFP, the green fluorescent protein, onto other cellular proteins; it is similar to forcing the proteins to hold a glow stick. That trick gave Scientists a simpler way to trace the movements of proteins under a microscope - without using synthetic dyes that are expensive. The novelty blazed across the field of biological imaging. In the year 2007, the mixing of GFP and two other fluorescent proteins allowed Scientists to paint mouse neurons a range of vivid colors in a method referred to as the "Brainbow." A year later, three Scientists, including the late Roger Tsien, an HHMI investigator, won the Nobel Prize in Chemistry for the discovery and development of GFP.

However, GFP has some disadvantages too. It is a comparatively clunky molecule built out of the restricted set of natural amino acids. Thus, GFP is not always bright enough to expose what Scientists are attempting to see.

As a consequence, Researchers turned back to Chemistry. Scientists had developed novel techniques to label cellular contents and cutting-edge microscopes, Lavis says; however the dyes for marking molecules within cells were still stuck in the nineteenth century. His team concentrated on rhodamines, because they are particularly cell-permeable and bright - so they slip into cells effortlessly and make them glow.

However, in spite of working with rhodamines for more than a century, only a few dozen colors have been created by the Chemists, of which most were similar shades varying from green to orange.

Making new rhodamines was not easy until recently. Scientists still used methods from the earliest days of chemistry, boiling chemical ingredients in sulfuric acid. This forces the molecules to associate together in what is known as a condensation reaction. Mixing in different building blocks can produce unusual and novel dyes. Ingredients still had to be tough enough in order to survive the boiling acid bath - which did not leave a lot of options.

Make it glow

Lavis's team, in the year 2011, developed an innovative method to tinker with rhodamines' structure, under milder conditions. Employing a reaction sparked by the metal palladium, the Scientists could skip the acid step and produce dyes with more intricate building blocks than had been employed earlier.

This gentler, kinder approach paved the way for a wide new world of dyes, and Lavis's team dove in. After four years, they launched the Janelia Fluor dyes, fluorescent molecules which are more stable too and are up to 50 times brighter when compared to other dyes. A tiny square-shaped appendage known as an azetidine ring is the secret behind the Janelia Fluor dyes. This structure was made possible only by Lavis's innovative chemistry approach.

There are a range of strategies that Scientists can use to get the bright dye molecules onto the protein they want to investigate. Subsequently, they can zero in on the lit-up protein, and watch it wiggle around and interact with other molecules - without the typical background fuzziness.

For us, it was a total revolution in the field of single-molecule imaging.

Xavier Darzacq, Molecular Biologist, the University of California, Berkeley

Before using the Janelia Fluor dyes, the fluorescent-tagged transcription factor proteins his team explored were very dim to record in crisp images. Researchers had to hold the camera shutter open for 10 ms to collect sufficient light. That is long enough for proteins to move about, and thus resulting in a blurry image - like a photograph of a squirmy toddler. Darzacq says that the Janelia dyes are bright enough that his team can record molecules in action in just a millisecond. Such quick snapshots have enabled his team to perform lab experiments he describes as, "simply unthinkable a few years ago."

Lavis's group has now found out how to fine-tune their fluorescent dyes, by tweaking rhodamines' structure even more. Rhodamines feature a basic four-ringed design with groups of atoms protruding from varied parts of the rings. In earlier work, the Scientists developed strategies for coarse tuning dyes – If an entire appendage is snipped out here, one can make a green dye. Or if a silicon atom is popped in there, one can get red. Lavis figured that by cautiously placing just a few new atoms in the dye structure, the chemical and color properties of the dyes could also be fine-tuned, enabling several shades of green from a single scaffold. It is like going from the characteristic eight pack of crayons to the jumbo box of 64.

The team explained a way to modify the dye structure's bottom ring, in a separate paper, published August 9th, 2017 in the journal ACS Central Science.

The key thing is that it's all modular and rational.

Luke Lavis, Group Leader, the Howard Hughes Medical Institute's Janelia Research Campus, Ashburn, Virginia

Lavis explains that when the right atoms are selected, the chemists can then design dyes with almost any property they want.

His team grafted several chemicals onto rhodamines, and then analyzed the new dyes' properties. "No one had ever looked at rhodamines in this kind of systematic way before," says Lead Co-author Jonathan Grimm, a Senior Scientist at Janelia.

Lavis says that in addition to inexpensive ingredients, only a single step was required to synthesize the dyes. That makes the dyes cheaper when compared to commercial alternatives - pennies per vial. His group is now able to share their work with Scientists all over the world due to the low cost. Lavis, Grimm, and colleagues have currently shipped thousands of vials to hundreds of different labs.

Ethan Garner, a Bacterial Cell Biologist at Harvard University says "These dyes are a complete game-changer." He has used them for tracing the path of single molecules in his lab. One major drawback was that the scientists did not have a lot of different colors to choose from. However, he now says, with Lavis's work, "They can actually cover the whole spectral range."

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