In 2004, Eric Betzig was unemployed, tinkering in his friend Harald Hess’s living room. In 2005, he was a research group leader at the impressive Janelia Research Campus in Virginia on his way to winning the 2014 Nobel Prize in chemistry. The tinkering had paid off, leading Betzig to invent a microscope that saw things no other microscopes could.

Betzig, who graduated from Ann Arbor’s Pioneer High School, spoke at the Medical School on Thursday morning about developing the photo-activated localization microscope, or PALM, and gave an overview of the recent developments in microscopy. 

Traditionally, the limiting factor for microscopes was not their magnification. Instead, it was a property called “resolution,” which refers to the shortest distance between two separate points in a microscope’s field of view that can still be distinguished as distinct entities. If you imagine a microscope as drawing a picture, microscopes with poor resolution draw with dull crayons while microscopes with good resolution draw with fine-tipped pencils.

As microscopes were improved, they hit a limit: The resolution could never get better than half a wavelength of light. Since microscopes use light to see, they can’t tell the difference between two objects closer together to each other than a ray of light. Because of this, microscopes could easily see cells and some of the larger objects inside. But important interactions such as viruses’ infecting a cell or the aggregating of proteins that cause Alzheimer’s were seen only very blurrily, if at all.

Betzig, however, found a way around this physical constraint through PALM.

PALM is a type of fluorescent microscopy. In fluorescent microscopy, the microscope doesn’t actually see the components of a cell, but instead glow-in-the-dark, or “fluorescent,” molecules that attach themselves to the features of interest. The problem when attempting to look at a super-high-resolution image is that each fluorescent molecule looks like a fuzzy blob — Betzig calls these “fuzzballs” — and these blobs start to overlap each other.

Betzig found a way through this problem by using fluorescent molecules that don’t automatically glow, but instead only glow when exposed to a certain color of light — hence the “photo-activated” component of PALM. He found a way to ensure that only a handful of these molecules would glow in a given image.

After taking many images, with only a few different molecules glowing in each one, he would clean the images so only the innermost cores of the fuzzballs were visible. Then, all of the images would be overlaid and the fuzzballs, now just bright points of light, would no longer be overlapping, thus surpassing the theoretical limit for resolution. Suddenly, a whole world of intracellular interactions was visible.

Despite the novelty and power of this approach, Betzig described becoming quickly tired of PALM.

“And so, again, I worked on PALM until 2008 and by that time I was sick of it for two reasons. One is that super-resolution became a big bandwagon with lots of people getting into it. And usually when that happens I want to run as far as possible in another direction. The other thing is that, like my earlier super-resolution methods, it has a lot of really intrinsic limitations,” Betzig said.

The limitations mainly revolved around the fact that PALM is optimized for cells on a slide and isn’t adept at taking three-dimensional videos.

“Basically, I got sick of looking at dead things,” Betzig said.

In 2008, he was inspired by his colleague Mats Gustafsson, who developed structured illumination microscopy, or SIM. This technique also beat the theoretical resolution limitation, but only by an order of two instead of the almost unlimited resolution achieved by PALM. For this reason, Gustafsson was not also considered for the Nobel Prize, a fact that Betzig takes issue with.

“Now, that was the reason the Nobel Committee stated — because it was only a factor of two instead of theoretically unlimited by … PALM — as to why SIM was not part of the Nobel Prize. But, in my opinion, that’s a tremendous mistake. Honestly, I feel that SIM is the technique that has answered and will continue to answer many biological questions,” Betzig said.

Now, Betzig works mostly with SIM and another microscopy technique called lattice light-sheet microscopy. Despite the quickly evolving nature of his field, he says that the important reminder is to use these technologies for practical purposes.

“Winning a Nobel Prize is fine. Publishing a bunch of cute papers is fine. But it all means diddly-squat if these tools don’t answer biological questions. And I’m really concerned about that. And it’s very important to me to make sure that these tools get into the hands of biologists.”

Betzig said many challenges remain. Preparing samples properly is more difficult than ever, and the huge amount of data coming out of the microscopes can be overwhelming.

“The problem with this microscope is each biologist comes for a week and leaves with 10 terabytes of data. And they have no fucking clue how to deal with it. You’ll never hear from them again because they’re too embarrassed to ask what to do,” he said.

Betzig hopes, however, that as technologies and information sciences advance, soon his main goal will be achieved.

“Putting these pieces together will get us closer than ever before to studying cell physiology as it actually happens.”

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