Making the invisible visible

As a teenager in the 1960s, Martin Chalfie formed an image in his mind of what it meant to be a scientist.

One half-century and a Nobel Prize later, he confessed last week to a Lehigh audience, many of the elements of his youthful imagination seem almost comical.

A scientist, thought the young Chalfie, would be a genius with innate scientific ability. He would conduct experiments that always worked the first time. He would use a peculiar way of thinking known as the scientific method.

He would work alone, and he would be a white male.

His journey since then, Chalfie said, has contradicted most of his early assumptions.

This was especially true in 2008, when Chalfie won the Nobel Prize for chemistry for introducing the Green Fluorescent Protein (GFP) as a biological marker. The achievement came after many failures.

And it was not a solo affair, as Chalfie shared the prize with Osamu Shimomura, a Japanese organic chemist, and Roger Tsien, an American biochemist.

The history of a biological marker

Chalfie, a professor of biological sciences at Columbia University, related the story of the Nobel Prize-winning research into GFP in a talk titled “GFP: Lighting up Life” on Wednesday, Feb. 11. The following day he gave a seminar titled “Mechanosensory transduction and its modification in Caenorhabditis elegans.”

His two-day visit to Lehigh was sponsored by Lehigh’s HHMI (Howard Hughes Medical Institute) program through the department of biological sciences.

It was in 1961, Chalfie said, that Shimomura first isolated GFP from a jellyfish. He had spent an entire summer grinding up and examining thousands of jellyfish without success.

One day, feeling frustrated, Shimomura threw all his prep in the sink. As he turned off the lights and prepared to leave the lab, he noticed the sink was glowing. The sink contained seawater, which contains calcium. Shimomura realized that by adding calcium, he could get his prep to glow, which led him to discover GFP.

Dropping experimental materials on the floor, on a counter or in a sink, Chalfie joked, “is actually a tried-and-true method in science.”

The idea to use GFP as “a lantern” occurred to Chalfie nearly three decades later, during a 1989 seminar about Shimomura’s work.

“I realized that if you could put this protein anywhere,” he said, “then all we’d have to do was shine a blue light on it and we’d see green. I got so excited that I ignored the rest of the seminar. I spent the entire hour fantasizing about what we could do with this molecule.”

GFP was of particular interest to Chalfie at that time because he was studying gene expression in the nematode C. elegans, a transparent worm that he had been investigating for 12 years.

“We were working on cloning the genes needed for touch sensitivity,” Chalfie said. “Once we cloned the gene, the first questions we wanted to address were: Where was the gene active? What cells turned on that particular gene? And we particularly wanted to know what touch-sensing cells expressed that gene.”

A variety of ways of examining gene expression had already been developed, Chalfie said, including antibody staining, B-galactosidase activity and in situ hybridization. But these methods required a lot of work and killed the specimen. Moreover, they did not provide a dynamic view of gene expression, merely “a snapshot in time.

“If you wanted to see what happened over time,” he said, “you had to prepare samples at different times and hope that you’d be able to see things.”

In contrast, he said, using GFP to study gene expression has many advantages: Its DNA is inherited; it does not hurt the organism because it is noninvasive; its small size allows it to fill the cell completely; and it enables researchers to observe living cells and organisms.

In 1992, after overcoming many obstacles—a missed connection, lack of equipment and naysayers in the science community—Chalfie successfully used GFP as a biological marker. His findings were published in the February 1994 issue of Science.

GFP’s influence

The discovery of GFP has made it possible to study processes that were previously invisible, leading to possible new treatments for diseases. It has facilitated the detection of contaminants in air and water.

The protein has also played a role in more than 160,000 scientific papers, Chalfie estimated.

“My friend Tony Hyman in Dresden,” he said, “discovered that there are some organelles that were not even known to exist in the cytoplasm and that seem to act like water droplets and fuse with each other and come apart. The only way you can see these organelles is [with] GFP.”

The other uses of GFP, Chalfie said, fall into three categories: fundamental, such as cell and tissue isolation, mutational analysis and brain circuitry; applied, such as addressing issues in medicine and health, drug discovery and making biosensors; and unexpected, such as new research in areas like optogenetics, finding land mines and making fluorescent silk from GFP moths.

The protein’s impact was amplified by Tsien, who extended the color palette of GFP. The technology was also employed by Harvard University neurobiologists Jeff Lichtman and Josh Sanes, who labeled the neurons in the brains of mice, resulting in a spectacular display known as Brainbow.

Lessons from GFP

GFP has taught him many valuable lessons, Chalfie said. Scientific success comes in many ways, he said, and many discoveries are accidental. Stubbornness and persistence are important. Scientific progress is cumulative, and university and grant support are essential, as is fundamental research. And all life should be studied, not just model organisms like C. elegans.  

Along the way, Chalfie realized that while some scientists are rightfully considered geniuses, genius itself is not a requirement for success. While Tsien won first prize at the Westinghouse Science Talent Search at the age of 16, Chalfie said, his own college grades were unimpressive.

Chalfie said he considers a “passion for the science, excitement for the work, being mystified and intrigued by puzzles that we approach every day” to be the essential qualities of a scientist.

At the end of his lecture, Chalfie showed a short film that emphasized the importance of basic research. The film, “Funding Basic Research to Revolutionize Medicine,” won the 2013 FASEB (Federation of American Societies for Experimental Biology) Stand Up for Science Competition. It illustrates how research funded in the 1960s on how bacteria protect themselves against viruses has led to many useful discoveries, including treatments for diabetes and strokes and medications for cancer and HIV.

Chalfie was introduced to his Lehigh audience by Alan J. Snyder, vice president and associate provost for research and graduate studies.

“The most important people in our lives,” said Snyder, “are often those who help us to see. So many of our aspirations and searches for understanding involve ways to see things that would otherwise escape us. Sometimes it involves literally making the invisible visible.”

During his time at Lehigh, Chalfie held six meetings with groups of faculty members from the department of biological sciences, the department of chemistry and the bioengineering program. He also met with graduate students and was the special guest at a reception and dinner at the President’s House.

Story by Rosa Rojas

Photo by Christa Neu