Beyond the diffraction limit


Rebecca Pool

Monday, March 30, 2015 - 14:00
Microscopy and Analysis talks to Nobel Laureate, Professor Eric Betzig, about his life, research and more.
Ask Professor Eric Betzig what he feels about his Nobel Prize in Chemistry and you might be surprised. "It's been very weird to see people's reaction as you know you're the same guy, but you're not treated as if you are," he tells Microscopy and Analysis.
"Still it settles down and I'm getting to the point now where I can go at least a day maybe without thinking about it," he adds. "In terms of drive, I don't see much to be gained by having it, so I hope soon I will rarely think about it."
But luckily for the world of microscopy, the reluctant Nobel Laureate has never been a reluctant scientist. His passion for science started with space, and growing up in the 1960s in the college town of Ann Arbour, the Apollo programme fuelled astronaut aspirations while friends and teachers linked to the University of Michigan stoked scientific curiosity.
"In Fourth grade, my teacher's husband was an assistant professor just when quarks were discovered," he says. "I remember writing letters to him, asking questions like 'What's the charge of a quark?', so there was always an outlet for my interest."
Come the end of school and with astronaut ambitions put aside, Betzig wanted to be a theoretical astrophysicist, so headed out to the California Institute of Technology (Caltech) to study physics.
However, a couple of summer placements at Caltech, setting up a system to explore instability modes in gas jets, would change his mind.
"This was my first real taste of doing experimental research and it was going to be very important for me," he says. "Before I'd thought 'well I'm going to be an astrophysicist' but by the time I was doing this, I was thinking 'Gee I really prefer doing stuff with my hands'."
So with his degree also in his hands, Betzig set out to study applied physics. It was the mid-1980s and only two universities offered the right Masters programmes; Cornell and Stanford.
Betzig chose Cornell - he'd had enough of California -  and plumped for a near-field microscopy project.
Comparison between diffraction-limited, summed TIRF (left) and PALM (right) images of a HFF-1 fibroblast. Images were rendered from 40,000 single-molecule images. More than 100,000 molecules are plotted in each PALM image. [Betzig]
The aim was to develop an optical microscope that could image living cells with the resolution of an electron microscope, in Betzig's words: a 'really big deal'.
His supervisors, Professors Mike Issacson and Aaron Lewis, had been using electron beams to drill 30 nm holes in opaque membranes to form apertures that could create a sub-wavelength light-source for scanning samples to generate super-resolution images. 
Betzig soon discovered the apertures were too fragile, so pioneered a scheme based on glass micropipettes previously developed by Erwin Neher and Bert Sakmann for use as recording electrodes to study single ion channels in cells.
Betzig and colleagues would pull glass micropipettes and then coat the structures with aluminium to create an opaque tapered probe with a sub-wavelength aperture.
Neher and Sakmann would later win a Nobel Prize in Physiology or Medicine for their micropipette-aided research, while Betzig would build a near-field scanning optical microscope, break the diffraction limit and take the technology to Bell Labs.
"The microscope was a pain in the neck to work with and the resolution gain over the diffraction limit wasn't huge, but it was enough to get me into the door of Bell Labs," he says. "So here I developed the technology to actually work routinely and with better resolution."
Reaching super-resolution
At Bell Labs, he switched the micropipette probe for an adiabatic tapered optical fibre that guided light more efficiently to the tip region and also invented a dithering technique to oscillate the probe and regulate its contact with a sample surface.
The probe was some ten thousand times brighter than past probes and could reach 15 nm resolution.
Come the early 1990s, Betzig had demonstrated super-resolution photo-lithography, nanoscale spectroscopy, and crucially, super-resolution fluorescent imaging of actin filaments in fibroblast cells.
"There was a couple of significant innovations to make the technology work at a better resolution and routinely as opposed to the pain-in-the-neck instrument I had at Cornell," he says. "But I was the first to see super resolution fluorescence imaging of cells and from these images it was clear that the signal and noise levels were good enough to see single fluorescent molecules from the near-field."
So he re-arranged his experimental set-up, and in just an afternoon, was able to repeatedly detect and determine molecule position at room temperature.
Eric Betzig: "I found PALM too slow... and it required ridiculously high-labeling densities to get high resolution." [Stephen Voss/HHMI]
What's more, he could also use the method to map the electric field distribution in his near-field aperture with molecular spatial resolution. He had achieved single molecule microscopy.
"This is probably one of the experiments I am most proud of," he says. ""We could see the molecules, determine their orientations... So we wrote up a paper in a week, it was accepted by Science in two weeks, and two weeks after that was in print."
Quantum beginnings
At the same time, Betzig was also working with his now lifelong friend, Harald Hess. Hess was pioneering low temperature STEM, so the pair combined Betzig's near-field probe with Hess's STEM to study luminesence centres in quantum wells.
"One of the reasons my boss at Bell Labs hired me was to find a way of looking at the energy levels in semiconductor structures at very high spatial resolution," explains Betzig. "After five or six years, Harald and I bowed to this idea, and we were surprised at the results."
The pair discovered the luminescence glowed from discreet points and in different wavelengths according to quantum well width.
And as Betzig highlights: "Even though there might have been many of these structures under our probe at any one time, we could still study them individually as they glowed in different colours."
So now having the ability to see single molecules and isolate discreet luminescent emitters, the seeds of Betzig's Nobel Prize winning breakthrough - super-resolution photoactivated localization microscope PALM - were sown.
Change of heart
But despite progress Betzig was tired of research. He'd realised near-field microscopy, given its short depth of focus, would never image live cells with the resolution of an electron microscope.
So come 1994, he left Bell Labs to work for his father, at the Ann Arbor Machines company, which manufactured parts for the automobile industry.
"I was full of myself from my success with near-field and thought I could change this [industry]," he adds. "My Dad's company was making enough money to support an idiot like me to mess around with ideas so he humoured me and I went there."
Yet Betzig still had research on his mind and within a year had published a short paper in Optics Letters ideas outlining a method for molecular optical imaging based on his Bell Labs' single molecule localisation research and luminescence results.
"This was the idea for PALM but I knew it would be a very challenging experiment," he says. "It would have been very difficult for biology at the time."
Betzig spent six years at his father's company, after which time the automobile industry has dwindled, but his scientific aspirations hadn't. Crucially, during his time in industry, Betzig had also learned, in his words, a huge lesson.
"The most painful and important lesson, and this is for business or science, if you want to make a significant impact you have to listen to the customer," he says. "Now, open any issue of Optics Express, Optics Letters and you see a tonne of microscopy stuff, but most of it, even if it's successful is not anything anybody really cares about. "
So with the customer in mind, Betzig left his father's company, started reading scientific literature, and learned green fluorescent protein had been discovered.
He quickly realised the potential of photoactivated fluorescent proteins in super-resolution microscopy, the impact this could have on biologists and reconnected with Hess.
Based on their work at Bell Labs and Betzig's most recent paper - now nearly a decade old - the pair built the first super-resolution photoactivated localization microscope (PALM) prototype in Hess's home.
The making of PALM
The method relies on the ability to turn the fluorescence of molecules on and off. 
Molecules are imaged many times, with a small subset of fluorescent tags switched on each time. These images can then be superimposed to yield a single picture crammed full of glowing molecules.
Eventually, Betzig and colleagues would compile thousands of images to generate a super-resolution image, but as the Nobel Laureate points out: "We had no cell culture facilities, molecular biology or cloning capabilities in Harald's living room."
So they tracked down Jennifer Lippincott-Schwarz and fellow biologists working with photoactivated GFP, at Florida State University, and persuaded them of their microscope's potential.
"Many people would have blown off these guys who were ten years from having written a paper and talking crazy talk," laughs Betzig. "But Jennifer said the microscope sounded great."
In 2005, Betzig and Hess moved to the National Institutes of Health campus and would soon start long, prosperous research careers at Janelia Farm Research Campus, Howard Hughes Medical Institute.
Working in Lippincott-Schwarz's laboratory, the researchers set to work developing PALM and within months were getting results, creating, for example images of organelles at the molecular level.
Moving on
But come 2008 Betzig was, as he puts it, 'bored and frustrated' to the same extent as he had been with his near-field microscopy in 1994.
As he points out: "I found PALM too slow, too damaging to look at much in the way of living things and it required ridiculously high labelling densities to get high resolution."
"The technologies are now only just developing to get to the kind of density that you really want to work with on a routine basis," he adds.
However, at around this time, microscopy pioneer, Ernst Stelzer, was fast developing light sheet microscopy to image fixed and live embryos. Here, a laser light sheet is repeatedly swept across the field of view of a sample to build a 3D image.
Betzig had heard about this technique but quickly noted that the Gaussian beams being used were too thick to look inside a single cell.
Drawing from the near-field microscopy research of his early days, he realised he could switch the thick Gaussian beam for a non-diffracting narrow Bessel beam to create a thin virtual light sheet to image inside single cells.
Driving light-sheet microscopy forward; Protozoan T Thermophila at eight consecutive time points [Betzig Lab]
By 2011, Betzig had perfected Bessel beam plane illuminating microscopy for live cell imaging, yet still felt he could achieve more.
To reduce the time taken to scan the section, he decided to divide the Bessel beam into parallel parts, pioneering a new class of light beams, the 2D optical lattice.
As Betzig explains, the lattices are created by modulating laser light with a fast-switching spatial light modulator, which are then filtered and focused together to form the light sheet.  
Crucially, these ultra-thin lattice light sheets can non-invasively image live specimens at higher spatial and temporal resolutions.
Looking to the future
Late last year, Betzig's team revealed videos of a host of processes from embryonic development in nematodes and fruit flies down to chromosomes moving in dividing cells, publishing results in Science.
And now the researcher is looking to integrate adaptive optics he has been working on for several years to the lattice light sheet process.
Neurons in the brain of a developing zebrafish: top: with adaptive optics showing an increase in signal and recovery of resolution; btm: without AO [Kai Wang, Eric Betzig, Janelia]
"This is only applicable to transparent organisms, but that's ok as there's still plenty we can learn biologically from those types of specimens," he says.
So with Nobel Prize in tow, research clearly continues for Betzig. But what, does he believe, is the secret of success? His answer is simple.
"Talk to people to find out what makes a difference,"he says. "And once you've convinced yourself it can make a difference, be passionate about it and work ridiculously hard."
Lead image credit: Stephen Voss/HHMI
Website developed by S8080 Digital Media