Never say never, because humans are clever. Physics teachers used to say that humans will never be able to see atoms through any microscope due to a fundamental barrier to resolution called the diffraction limit. But like the four-minute mile and the sound barrier, this record was made to be broken. Today, optical engineers are doing it. Individual atoms are coming into focus!
Conventional microscopy techniques, namely the confocal microscope or deconvolution processes, are resolution limited, ~250 nm, by the diffraction properties of light as developed by Ernst Abbe in 1873. This diffraction limit is appreciably above the size of most multi-protein complexes, which are typically 20–50 nm in diameter. In the mid 2000s, biophysicists moved beyond the diffraction barrier by structuring the illumination pattern and then applying mathematical principles and algorithms to allow a resolution of approximately 100 nm, sufficient to address protein subcellular colocalization questions. This “breaking” of the diffraction barrier, affording resolution beyond 200 nm is termed super resolution microscopy. More recent approaches include single molecule localization (such as PhotoActivated Localization Microscopy (PALM)/STochastic Optical Reconstruction Microscopy (STORM)) and point spread function engineering (such as STimulated Emission Depletion (STED) microscopy). [Emphasis added.]
Super-resolution microscopy has exploded since the diffraction limit was surpassed. Instead of 100nm, scientists are imaging objects at 10nm and smaller. Each new paper pushes the limit: down to 5nm, 4nm, 3nm, where individual domains of molecular machines can be seen.
These successes are largely due to the introduction of cryo-electron microscopy (see paper in Protein Science), which shock-freezes the specimen to stabilize it before imaging it in an electron microscope. The technique earned three biophysicists the Nobel Prize in Chemistry in 2017. Conceived of in the late 1990s, cryo-EM went mainstream about 2013 and is now state of the art. Even so, cryo-EM had its own resolution barrier.
In October, the Max Planck Institute announced a new world record for cryo-EM by Holger Stark that broke through the barrier. “For the first time, his group succeeded in observing individual atoms in a protein structure and taking the sharpest images ever with this method.” Now, scientists will no longer be measuring in nanometers but in Angstrom units — an order of magnitude smaller.
Using the new microscope, the scientists have taken more than one million images of the protein apoferritin to map the molecular structure with a resolution of 1.25 angstroms. One angstrom is equivalent to a ten millionth of a millimeter. “We now visualize single atoms in the protein — a milestone in our field,” explains structural biologist Stark. “For us, it was like putting super glasses on the microscope. The new structure reveals details never seen before: We can even see the density for hydrogen atoms and single atom chemical modifications.”
Nature News and Views comments that “This unprecedented feat would not have been thought feasible merely a decade ago.” The technique used by Stark’s team (published in the same issue of Nature by Yip et al.) is called “single particle cryo-EM.” It’s part of a “resolution revolution” that began in 2013. Stark’s team imaged a ferritin molecule, a benchmark for cryo-EM because of its stability and symmetry, but a companion paper in Nature by Nakane et al. describes their success at imaging a portion of a more motile protein complex at 1.7 angstrom resolution. This approaches the length of a carbon-carbon bond (1.5 angstroms, according to Phys.org).
The developments in cryo-EM hardware described by Yip, Nakane and their respective colleagues have driven a major advance in the resolution of single-particle cryo-EM. Each team used hardware that tackled distinct aspects of cryo-EM imaging that had previously limited the resolution attainable. With these technologies, the increased signal-to-noise ratio of cryo-EM images will expand the technique’s applicability. For example, this might include using the technique to determine high-resolution structures of heterogeneous samples such as those formed of membrane proteins, or macromolecular complexes that vary in conformation or composition. Perhaps the melding of these technologies will enable the determination of cryo-EM structures at a resolution beyond even 1 Å. This once might have seemed a near impossible quest to embark upon.
More Imaging Advances
Adaptive Optics has long been used by astronomers to sharpen images of stars by reducing the smearing caused by turbulence in the atmosphere. The technique uses a laser beam as a guide, and rapidly shifts the telescope’s mirror to keep the reference beam steady. Can that technique be used at the other end of the tube — i.e., in a microscope? Yes! A team led by Daniel Hammer of the Food and Drug Administration (FDA) was able to image tiny microglia in the inner limiting membrane of the human retina using adaptive optics (AO). Combining this with optical coherence tomography (OCT), which allows optical depth sectioning of images for a 3-D view, they were able to follow these microglia, the “first responders to neural injury,” in real time in live human subjects. That is only one of many possible applications of this method, they say; “the ability to visualize macrophage cells without fluorescent labeling in the live human eye represents an important advance for both ophthalmology and neuroscience, which may lead to novel disease biomarkers and new avenues of exploration in disease progression.” The results were published last month in PNAS.
More about super resolution imaging can be found in the Journal of the American Chemical Society: “Super-resolution Microscopy with Single Molecules in Biology and Beyond — Essentials, Current Trends, and Future Challenges.” Authors Leonhard Möckl and W. E. Moerner of Stanford say, “Despite the tremendous progress, the full potential of single-molecule super-resolution microscopy is yet to be realized, which will be enabled by the research ahead of us.”
Multifocus 3-D Imaging took a leap with a new technique that Phys.org calls a “simple way to capture high quality 3-D images of live cells and organisms.” A team led by Sheng Xiao of Boston University used a z-splitter prism to split light beams from the specimen into “several images, each focused to a different depth in the sample, in a single camera frame.” Instead of a flat image at one focal plane, this technique yields 3-D images from standard optical microscopes. “We used a z-splitter prism that can be assembled entirely from off-the-shelf components and is easily applied to a variety of imaging modalities such as fluorescence, phase-contrast or darkfield imaging,” said Xiao. It can be “simply added to most existing systems and is easy to replicate, making it accessible to other researchers.” Their technique is described in the journal Optica.
Great Tools for ID
Needless to say, these advances in imaging will be a boon for design science. Instead of being restricted to the hazy image of the bacterial flagellum available to Michael Behe in 1996, scientists can now look in detail at individual molecules making up the machine. Many journal papers are already describing several iconic molecular machines in unprecedented detail. Here are some recent examples:
Mitochondria. Austrian scientists resolved the coupling mechanism in the mammalian respiratory Complex I (the first machine in the mitochondrion’s electron transport chain) at 2.3 angstroms (Kampjut and Sazanov, Science). “The resolution of some structures was sufficient to see water molecules and to trace putative paths for proton transfer within the proton-pumping membrane domain,” a summary paragraph says. “The structures add valuable details that provide a basis for generating mechanistic hypotheses for this crucial complex.” Visual proof of cryo-EM’s value can be seen in two movies they made, available in the Supplementary Materials.
Ribosome. How molecular machines are assembled is of great interest to design science. The ribosome, which translates mRNA into protein, consists of a small 40S subunit and a large 60S subunit. These come together into a working machine with the aid of 200 factors in “a highly coordinated manner,” say Michael Ameismeyer et al. in Nature. Using cryo-EM, the team was able to figure out the “structural basis for the final steps of human 40S ribosome maturation.”
ATP synthase. Another favorite ID icon has been imaged with cryo-EM in enough detail to see how the peripheral stalk is assembled. The team of Jiulia He included Nobel laureate John E. Walker and has been published in PNAS. Assembly of human ATP synthase requires 27 nucleus-encoded proteins and two others encoded in the mitochondria. Super resolution microscopy now allows scientists to see “how components of the peripheral stalk and three associated membrane subunits are assembled and introduced into the enzyme complex.” Another paper in Nature Structural & Molecular Biology reveals “Cryo-EM structure of the entire mammalian F-type ATP synthase.”
Bacterial flagellum. How could we not mention the molecular machine that opened the eyes of many to intelligent design? New results using cryo-EM technology reveal details about the flagellum that Chang et al., in Nature Structural & Molecular Biology, used cryo-EM to propose a “Molecular mechanism for rotational switching of the bacterial flagellar motor.” In the team’s surprising model, the rotor is set in motion by other rotors! A ring of MotA subunits turns the rotor like a set of gears, and can rapidly switch to turn the other way.
Commenting on this model in Nature Structural & Molecular Biology, Keiichi Namba described it as a “two-cogwheel” arrangement. This might explain how the rotor can switch directions in a quarter turn, as Jed Macosco has said. Namba says,
The CW rotation of MotACD could generate the torque for C ring rotation in either CCW or CW directionsthrough different interaction modes with FliG at the top of the C ring, that is, either with the inner side of the tube (closer to the motor axis) or with the opposite outer side… This is just like a two-cogwheel gear system, composed of small and large cogwheels, with cogs at the cylinder edges that can switch their relative positions; the small cogwheel (MotA) always generates torque in one direction, but it can mesh with the large cogwheel(FliG in the C ring) via either its internal or external edges, hence driving rotation of the large cogwheel in either direction.
These new high-res images should be eye candy to Behe and all other ID proponents. Imagine peering under the hood of this machine and observing the rotor and stator of this motor at high resolution. Namba was clearly impressed, saying that the flagellum operates at “almost 100% efficiency” and has been measured with a maximum speed of “1,700 revolutions per second” (102.000 rpm), “which is much faster than that of a Formula One racing car engine.”
The Closer You Look
Super resolution microscopy will certainly be a boon for design science. ID advocates have often pointed out that the closer one looks at biological systems, the more engineered they look. For now, the state of the art requires piecing static images into models of motion. Will scientists ever succeed at watching machines like the ribosome, ATP synthase, and the bacterial flagellum in real time, like movies, at super resolution? Remember, never say never, because humans are clever. We can already take great interest and delight in what the “resolution revolution” has brought us.