Electron Microscopy: New Type of Genetic Tag Illuminates Life in Never-Before-Seen Detail

Electron Microscopy: New Type of Genetic Tag Illuminates Life in Never-Before-Seen Detail

  11:01:00 PM    Science Lover

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By modifying a protein from a plant that is much favored by science, researchers at the University of California, San Diego School of Medicine and colleagues have created a new type of genetic tag visible under an electron microscope, illuminating life in never-before-seen detail.

Confocal fluorescence images of miniSOG-targeted 
endoplasmic reticulum (A), Rab5a (B), zyxin (C), 
tubulin (D), ²-actin (E), ±-actinin (F), mitochondria
(G), and histone 2B (H) in HeLa cells; scale bars, 
10 µm. (Credit: Xiaokun Shu, Varda Lev-Ram, Thomas 
J. Deerinck, Yingchuan Qi, Ericka B. Ramko, Michael W. 
Davidson, Yishi Jin, Mark H. Ellisman, Roger Y. Tsien.
A Genetically Encoded Tag for Correlated Light and 
Electron Microscopy of Intact Cells, Tissues, and 
Organisms. PLoS Biology, 2011; 9 (4): e1001041 
DOI: 10.1371/journal.pbio.1001041)

Led by Nobel laureate Roger Tsien, PhD, Howard Hughes Medical Institute investigator and UCSD professor of pharmacology, chemistry and biochemistry, a team of scientists radically re-engineered a light-absorbing protein from the flowering cress plant Arabidopsis thaliana. When exposed to blue light, the altered protein produces abundant singlet oxygen, a form of molecular oxygen that can be made visible by electron microscopy (EM).

The findings are published in the online, open access journal PLoS Biology.

Tsien was co-winner of the 2008 Nobel Prize in chemistry for his role in helping develop and expand the use of green fluorescent protein (GFP), a protein from jellyfish that is now widely employed in light microscopy to peer inside living cells or whole animals and observe molecules interacting in real-time. Tsien said the development of the small, highly engineered Arabidopsis protein, dubbed "miniSOG," may elevate the abilities of electron microscopy in the same way that GFP and its relatives have made modern light microscopy in biological research much more powerful and useful.


"The big advantage of EM is that it has much higher spatial resolution than light microscopy. You can get up to a hundred-fold higher useful magnification from EM than from light microscopy," said Tsien. The result has been extraordinarily detailed, three-dimensional images of microscopic objects at resolutions measuring in the tens of nanometers, tiny enough to meticulously render the internal anatomy of individual cells. But current EM technologies do not distinguish or highlight individual proteins in these images. Although individual proteins can be tagged with GFP or other fluorescent proteins to aid localization by light microscopy, there has been no equivalent technology for the higher-resolution images provided by EM.

To create this ability, the scientists began with a protein from Arabidopsis that absorbs incoming blue light. It's normal function is to trigger biochemical signals that inform the plant how much sunlight it is receiving. "We rationally engineered the protein based on its atomic model so that it changes incoming blue light into a little bit of green fluorescence and a lot of singlet oxygen," said the paper's first author, Xiaokun Shu, now an assistant professor at UC San Francisco. Established methods were then used to convert singlet oxygen production into a tissue stain that the electron microscope can "see." The scientists tested the modified protein's utility as an EM marker by first using it to confirm the locations of several well-understood proteins in mammalian cells, nematodes and rodents, and then used miniSOG to successfully tag two neuronal proteins in mice whose locations had not been known.

Tsien is optimistic that miniSOG will grant new powers to electron microscopy, permitting scientists to pursue answers to questions previously impossible to ask. MiniSOG will especially be useful to scientists who investigate cellular and subcellular structures including neuronal circuits at nanometer resolution in multicellular organisms since previous methods have great difficulty in achieving both efficient labeling and good preservation of the structures under study. While EM can provide much higher useful magnification than light microscopy, EM will not replace light microscopy. "When we use miniSOG, we see the tagged proteins plus the landmarks that we are used to navigating by," said Tsien. "On the other hand, EM has the disadvantage that it gives a snapshot of cells before we killed them (to make the image), whereas light microscopy can show the dynamics in live cells. Each technique has different complementary strengths and weaknesses."

Co-authors of the paper include: Varda Lev-Ram, UCSD Department of Pharmacology; Thomas J. Deerinck, National Center for Microscopy and Imaging Research, Center for Research on Biological Systems, UCSD; Yingchuan Qi and Yishi Jin, Howard Hughes Medical Institute, UCSD and UCSD Division of Biological Science; Ericka B. Ramko and Michael W. Davidson, National High Magnetic Field Laboratory and Department of Biological Science, Florida State University; and Mark H. Ellisman, National Center for Microscopy and Imaging Research, Center for Research on Biological Systems, UCSD and UCSD Department of Neurosciences

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Record-Breaking High-Res Optical Technique

Record-Breaking High-Res Optical Technique

  11:33:00 PM    Science Lover

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Conventional wisdom holds that optical microscopy can't be used to "see" something as small as an individual molecule. But science has once again overturned conventional wisdom. Secretary of Energy, Nobel laureate and former director of the Lawrence Berkeley National Laboratory (Berkeley Lab) Steven Chu led the development of a technique that enables the use of optical microscopy to image objects or the distance between them with resolutions as small as 0.5 nanometers -- one-half of one billionth of a meter, or an order of magnitude smaller than the previous best.

Graph on top shows that with the active feedback 
system off there is a resolution drift of about 0.3 pixels 
or 19 nanometers, but with the feedback system on 
resolution is maintained at better than 0.01 pixels, 
or about 0.64 nanometers. Image on bottom shows 
individual Cyanine (Cy) fluorescent dye molecules – 
Cy3 and Cy5 - used to label 20 base pairs of double-
stranded DNA. (Credit: Image courtesy of DOE/
Lawrence Berkeley National Laboratory)

"The ability to get sub-nanometer resolution in biologically relevant aqueous environments has the potential to revolutionize biology, particularly structural biology," says Secretary Chu. "One of the motivations for this work, for example, was to measure distances between proteins that form multi-domain, highly complex structures, such as the protein assembly that forms the human RNA polymerase II system, which initiates DNA transcription."

Secretary Chu is the co-author of a paper now appearing in the journal Nature that describes this research. The other authors are Alexandros Pertsinidis, a post-doctoral researcher and member of Chu's research group at the University of California (UC) Berkeley, who is now an assistant professor at the Sloan-Kettering Institute, and Yunxiang Zhang, a member of Chu's research group at Stanford University.

According to a law of physics known as the "diffraction limit," the smallest image that an optical system can resolve is about half the wavelength of the light used to produce that image. For conventional optics, this corresponds to about 200 nanometers. By comparison, a DNA molecule measures about 2.5 nanometers in width.

While non-optical imaging systems, such as electron microscopes, can resolve objects well into the subnanometer scale, these systems operate under conditions not ideal for the study of biological samples. Detecting individual fluorescent labels attached to biological molecules of interest using charge-coupled devices (CCDs) -- arrays of silicon chips that convert incoming light into an electrical charge, has yielded resolutions as fine as five nanometers. However, until now this technology has been unable to image single molecules or distances between a pair of molecules much less than 20 nanometers.

Chu and his co-authors were able to use the same CCD-fluorescence technology to resolve distances with subnanometer precision and accuracy by correcting a trick of the light. The electrical charges in a CCD array are created when photons strike the silicon and dislodge electrons, with the strength of the charge being proportional to the intensity of the incident photons. However, depending upon precisely where a photon hits the surface of a silicon chip, there can be a slight difference in how the photon is absorbed and whether it generates a measurable charge. This non-uniformity in the response of the CCD silicon array to incoming photons, which is probably an artifact of the chip manufacturing process, results in a blurring of pixels that makes it difficult to resolve two points that are within a few nanometers of one another.

"We have developed an active feedback system that allows us to place the image of a single fluorescent molecule anywhere on the CCD array with sub-pixel precision, which in turn enables us to work in a region smaller than the typical three pixel length-scale of the CCD non-uniformity," says Pertsinidis, who is the lead author on the Nature paper. "With this feedback system plus the use of additional optical beams to stabilize the microscope system, we can create a calibrated region on the silicon array where the error due to non-uniformity is reduced to 0.5 nanometers. By placing the molecules we want to measure in the center of this region we can obtain subnanometer resolution using a conventional optical microscope that you can find in any biology lab."

Chu says that the ability to move the stage of a microscope small distances and calculate the geometric center (centroid) of the image makes it possible to not only measure the photo-response non-uniformity between pixels, but also to measure the non-uniformity within each individual pixel.

"Knowing this non-uniformity then allows us to make corrections between the apparent position and the real position of the image's centroid," says Chu. "Since this non-uniform response is built into the CCD array and does not change from day to day, our active feedback system allows us to image repeatedly at the same position of the CCD array."

Pertsinidis is continuing to work with Chu and others in the group on the further development and application of this super-resolution technique. In addition to the human RNA polymerase II system, he and the group are using it to determine the structure of the Epithelial cadherin molecules that are responsible for the cell-to-cell adhesion that holds tissue and other biological materials together. Pertsinidis, Zhang, and another postdoc in Chu's research group, Sang Ryul Park, are also using this technique to create 3D measurements of the molecular organization inside brain cells.

"The idea is to determine the structure and dynamics of the vesicle fusion process that releases the neurotransmitter molecules used by neurons to communicate with one another," Pertsinidis says. "Right now we are getting in situ measurements with a resolution of about 10 nanometers, but we think we can push this resolution to within two nanometers."

In a collaboration with Joe Gray, Berkeley Lab's Associate Director for Life Sciences and a leading cancer researcher, postdocs in Chu's research group are also using the super-resolution technique to study the attachment of signaling molecules on the RAS protein, which has been linked to a number of cancers, including those of the breast, pancreas, lung and colon. This research could help explain why cancer therapies that perform well on some patients are ineffective on others.

In addition to its biological applications, Pertsinidis, Zhang and Chu in their Nature paper say their super-resolution technique should also prove valuable to characterize and design precision photometric imaging systems in atomic physics or astronomy, and allow for new tools in optical lithography and nanometrology.

This research was supported by the National Institutes of Health, the National Science Foundation, the National Aeronautics and Space Administration, and the Defense Advanced Research Projects Agency.

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Microscope 'Sees' Atoms for First Time

Microscope 'Sees' Atoms for First Time

  9:16:00 PM    Science Lover

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UCLA researchers report in the April 30 edition of the journal Cell that they have imaged a virus structure at a resolution high enough to effectively "see" atoms, the first published instance of imaging biological complexes at such a resolution.

Cover of the journal Cell showing an artistic 
representation of a Cryo-EM 3-D reconstruction 
of an aquareovirus, the colors are added to show 
contrast between various structures in the virus. 
(Credit: Image courtesy of UCLA)

The research team, led by Hong Zhou, UCLA professor of microbiology, immunology and molecular genetics, used cryo-electron microscopy to image the structure at 3.3 angstroms. An angstrom is the smallest recognized division of a chemical element and is about the distance between the two hydrogen atoms in a water molecule.

The study, the researchers say, demonstrates the great potential of cryo-electron microscopy, or Cryo-EM, for producing extremely high-resolution images of biological samples in their native environment.

"This is the first study to determine an atomic resolution structure through Cryo-EM alone," said Xing Zhang, a postdoctoral candidate in Zhou's group and lead author of the Cell paper. "By proving the effectiveness of this microscopy technique, we have opened the door to a wide variety of biological studies."

With traditional light microscopy, a magnified image of a sample is viewed through a lens. Some samples, however, are too small to diffract visible light (in the 500 to 800 nm range, or 5,000 to 8,000 angstroms) and therefore cannot be seen. To image objects at the sub-500 nm scale, scientists must turn to other tools, such as atomic force microscopes, which use an atomically thin tip to generate an image by probing a surface, in much the same way a blind person reads by touching Braille lettering.

With electron microscopy, another sub-500 nm technology, a beam of electrons is fired at a sample, passing through empty areas and bouncing off dense areas. A digital camera reads the path of the electrons passing through the sample to create a two-dimensional projection image of the sample. By repeating this process at hundreds of different angles, a computer can construct a three-dimensional image of the sample at a very high resolution.

Zhou is faculty director of the Electron Imaging Center for Nanomachines (EICN) at UCLA's California NanoSystems Institute, which is using cryo-electron microscopy to create 3-D reconstructions of nano-machineries, nano-devices and biological nano-structures, such as viruses.

Structurally accurate 3-D reconstructions of biological complexes are possible with cryo-electron microscopy because the samples are flash frozen, which allows them to be imaged in their native environment, and the microscope operates in a vacuum, because electrons travel better in that environment. The Cell paper focused on a structural study of the aquareovirus, a non-envelope virus that causes disease in fish and shellfish, in an effort to better understand how non-envelope viruses infect host cells.

"We are extremely excited about the recent breakthrough achieved by Hong Zhou and his team at the EICN lab," said Leonard H. Rome, senior associate dean for research at the David Geffen School of Medicine at UCLA and associate director of the California NanoSystems Institute. "The ability to understand the structure of viruses at an atomic level will open avenues for manipulating them for use in drug delivery and propel numerous innovations in treatments of diseases. UCLA is fortunate to have such specialized instrumentation and the expertise of Professor Zhou and his team to take advantage of these marvelous microscopes."

Viruses can be classed into two types: envelope and non-envelope. Envelope viruses, which include influenza and HIV, are surrounded by an envelope-like membrane which the virus uses to fuse with and infect a host cell. Non-envelope viruses lack this membrane and instead use a protein to fuse with and infect cells. This process was poorly understood until Zhou's study.

"Through better knowledge of virus structures, we hope to engineer medications in three ways," Zhou said. "If we understand how viruses work, first we can identify small molecules or drugs that block their infection; second, we can engineer ultra-stable and non-infectious virus-like particles as optimal vaccines; and third, we can alter their characteristics so that instead of delivering a disease, viruses could deliver medications.

"Indeed, we are working with UCLA physicians and engineers to engineer viruses for gene therapy and drug delivery," he said. "In essence, we hope to take advantage of millions of years of evolution that have made viruses incredibly effective delivery platforms."

From the high-resolution 3-D images produced with the cryo-electron microscopy, Zhou's group was able to determine that the aquareovirus employs a priming stage to accomplish cell infection. In its dormant state, the virus has a protective protein covering, which it sheds during priming. Once the outer shell has been shed, the virus is in a primed state and is ready to use a protein called an "insertion finger" to infect a cell.

The team's study ushers in a new era of structural biology for understanding important biological processes. The group was able to discover this functionality because of the accurate structural model produced through cryo-electron microscopy. In addition to producing a high-resolution 3-D image of samples, the technology allows samples to be imaged in their native environment, so the structural model is faithful to the original sample. From a technical point of view, this work also demonstrates the power of cryo-electron microscopy in obtaining 3-D structures of biological complexes without needing to grow a crystal.

The California NanoSystems Institute at UCLA is an integrated research center operating jointly at UCLA and UC Santa Barbara whose mission is to foster interdisciplinary collaborations for discoveries in nanosystems and nanotechnology; train the next generation of scientists, educators and technology leaders; and facilitate partnerships with industry, fueling economic development and the social well-being of California, the United States and the world. The CNSI was established in 2000 with $100 million from the state of California and an additional $250 million in federal research grants and industry funding. At the institute, scientists in the areas of biology, chemistry, biochemistry, physics, mathematics, computational science and engineering are measuring, modifying and manipulating the building blocks of our world -- atoms and molecules. These scientists benefit from an integrated laboratory culture enabling them to conduct dynamic research at the nanoscale, leading to significant breakthroughs in the areas of health, energy, the environment and information technology.

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Neuron Connections Seen in 3-D

Neuron Connections Seen in 3-D

  3:44:00 PM    Science Lover

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A team of researchers from the Max Planck Institute of Biochemistry, in Germany, led by the Spanish physicist Rubén Fernández-Busnadiego, has managed to obtain 3D images of the vesicles and filaments involved in communication between neurons. The method is based on a novel technique in electron microscopy, which cools cells so quickly that their biological structures can be frozen while fully active.

This three-dimensional visualization of synapses shows the tomography mail synaptic vesicles (yellow), cell membrane (purple), connectors between vesicles (red), filaments that anchor the vesicles to the cell membrane (blue), microtubule (dark green), material synaptic space (light green) and postsynaptic density (orange). (Credit: Fernández-Busnadiego et al.)

"We used electron cryotomography, a new technique in microscopy based on ultra-fast freezing of cells, in order to study and obtain three-dimensional images of synapsis, the cellular structure in which the communication between neurons takes place in the brains of mammals" Rubén Fernández-Busnadiego, lead author of the study which features on the front cover of this month's Journal of Cell Biology and a physicist at the Max Planck Institute of Biochemistry, in Germany, said.

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