How Light Microscopy Forever Changed Scientific Imaging
The Pittsburg Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon) is the premier annual laboratory science conference. The 66-year-old conference sets the tone for the rest of the year, with manufacturers debuting new analytical instrumentation that is sure to play a role in future research discoveries, along with lecturers delivering technical insights into their groundbreaking work, including how to propel the industry forward.
This year is no exception as Pittcon’s technical program features two Nobel Laureates (and others) speaking to the advent of super-resolution microscopy, and how it’s revolutionized scientific imaging in the last decade. W.E. Moerner and Eric Betzig comprise two-thirds of the team that was honored with the 2014 Nobel Prize in Chemistry for the development of super-resolved fluorescence microscopy.
Previously, the clarity and focus of optical microscopy was limited by the diffraction limit to features larger than about 250 nanometers (nm), which is about half the wavelength of visible light. While microscopists were trying to image very tiny objects inside a cell or other materials, they would still appear large because of optical diffraction. For example, a single point source would look like a giant 250-nm fuzz ball when, in actuality, the source was about 20 to 40 nm.
Although electron microscopy and X-ray technologies could surpass this level of detail, those techniques required scientists to kill a cell in order to make the observation—which is not very conducive to the research of biological processes.
By keying in on fluorescent light, however, Moerner and Betzig (working separately) were able to bypass this limit and lay the foundation for single-molecule microscopy. The method relies on the ability to turn the fluorescence of individual molecules on and off. Scientists image the same area multiple times, letting just a few interspersed molecules glow each time. Superimposing these images yields a dense super-image resolved at the nanolevel.
“My laboratory was the first to optically detect a single molecule,” Moerner explained to Laboratory Equipment. “Ever since then we have been exploring single molecules in every way we can think of. At the beginning, the experiments were liquid helium-based and related to optical storage. In the mid-90s, we moved to room temperature experiments, along with almost everyone in the field, and that’s when we could begin using these methods of single molecule imaging on cells and polymers.”
Moerner has helped reveal key details of how Huntington’s proteins damage the brain, how bacterial proteins regulate DNA replication and cellular division in time and space, and the precise structures of the cellular antennae that, if mutated, can trigger various diseases in humans, among other discoveries.
Currently, his lab at Stanford University still works with single molecules—both as a means to image biological structures and as objects to be observed and tracked for dynamics experiments, allowing the motion of a single molecule to indicate what is going on inside the cell.
Another major thrust in Moerner’s lab is the development of advanced ways to image single molecules in three dimensions. The Nobel Laureate and his team has already invented a number of new ways to achieve 3-D imaging using manipulation of the point-spread function of the microscope and manipulation of the fundamental response of the microscope using optical components outside the unit.
“Our main purpose throughout all of this is to be as quantitative as possible,” Moerner said. “We want to provide images that have the best precision and accuracy possible.”
The quantitative interpretation of super-resolution images is definitely a major focus of the field nowadays, agrees Bo Huang, an Associate Professor of Biochemistry and Biophysics at the University of California, San Francisco. Huang will be speaking about “Life Inside the Cell” at Pittcon as part of the Waters Symposium, which Betzig is headlining.
In addition to quantitative interpretation, Huang’s lab is also working on the application of super-resolution microscopy instrumentation to biological structure studies. Specifically, they are developing super-resolution and light-sheet microscopes that can visualize subcellular structures at a higher spatial resolution, record long-term cell behavior and track cells in intact animals.
“My take is once you can get a biological structure enabled with the proper process, that’s the time when you can convert the biological question into a physical question,” Huang explained to Laboratory Equipment. “That has been one of the aspects I’ve seen that is lagging behind a little, in order to connect the microscopy observation to a real, logical interpretation of the results.”
Like any young technique—or old for that matter—there are still bottlenecks and challenges persistent in super-resolution microscopy. Moerner points to the need for better fluorophores that emit more photons from each single molecule, while Huang laments the extended period of time it takes to actually image live cells and tissues.
While obviously pervasive in biological studies, there are some areas of research where super-res microscopy has the potential for greatness but has so far been underutilized, including polymers, gels and separation science, according to Moerner.
“My hope is that, technologically, super-res microscopy will become more mature and the lab will eventually use it as more of a standard method, like confocal microscopy,” Huang said. “It takes a lot of pretty boring effort to take a technique from something that is highly advanced in the lab to something that almost everyone can use. In the end, it might not be super-res microscopy that we can push to do live cell and tissue imaging. But, a lot of the concepts we have developed for super-res microscopy may merge with other methods to create the next generation of methods that overcome these challenges and ultimately understand how things behave in a live organism.”
