A case of “Back to the Future:” Promoting electron microscopy in Kentucky

There’s no doubt that technology has had a significant, wide-ranging impact on research as a whole.

Yet what if some research is best conducted with instrumentation and methodology developed decades ago? Such is the case with transmission electron microscopy, one of the key capabilities of Western Kentucky University’s Ogden College Electron Microscopy Center.

A proposed partnership between the University of Louisville, Western Kentucky University and the University of Kentucky hopes to revive the use of transmission electron microscopy through the creation of an electron microscopy core, which would not only help researchers and students complete their work, but could also solve one of the primary challenges of TEM: make it more cost-effective, thereby expanding its use.

Reversing a long-time decline

First, a quick electron microscopy primer (for more details, refer to the FAQs sidebar). Rather than using beams of light to examine a specimen, electron microscopes use beams of fast-moving electrons to examine carefully prepared specimens in an airless vacuum chamber.

Instead of lenses, electron microscopes use electromagnets to bend the electron beams, which produces magnification and a subsequent photograph (or electron micrograph) that captures the image.

WKU’s transmission electron microscope. Samples can be viewed directly on a phosphorescent screen (the green image) or on a computer monitor (on the right).

Several types of electron microscopes exist, including transmission electron microscopes, which are the focus of the proposed electron microscopy core. TEMs are valuable because they’re the most powerful type of electron microscopes and can be used to see things that are smaller than one nanometer.

Because TEMs are intended to view samples you wouldn’t otherwise be able to study with a typical microscope, their use requires thorough training and careful preparation.

“When using an electron microscope, your sample has to be thin enough so electrons can get through it, which isn’t simple because electrons have mass,” said John Andersland, Ph.D., director of Western Kentucky University’s Ogden College Electron Microscopy Center. “TEM samples are so thin that they’re essentially two-dimensional.”

And that’s one of the primary reasons that TEM use has dwindled over the last two or so decades. For example, TEMs were widely used in hospitals in the 1960s and 70s, but were then replaced with newer technology including CAT scanners.

Many universities—including the University of Louisville—saw their electron microscopy facilities close due to lack of use.

Yet TEM “remains the best (and sometimes only) way to address certain questions in the biomedical sciences, and different researchers need to use it, even if they would rather not,” Andersland said.

People and purpose

Led by Martha Bickford, Ph.D., professor, University of Louisville, and associate program director, Kentucky Biomedical Research Infrastructure Network (KBRIN), the electron microscopy core is intended to be a collaborative effort under the KBRIN umbrella, which would help expand the use of electron microscopy across the state and fill existing electron microscopy gaps.

While the KBRIN network contains excellent equipment for electron microscopy, most investigators don’t have the infrastructure or expertise in their own laboratories to prepare tissue for electron microscopic examination. As a result, it’s necessary to establish a core facility to aid both established and new investigators in sample preparation. The core would also provide standardized training for students and new investigators to use electron microscopes and interpret their images.

A copper grid with ultra-thin sections ready to view in the transmission electron microscope.

At the University of Louisville, many of the investigators interested in using electron microscopy are associated with past or present Centers of Biomedical Research Excellence (COBRE). Therefore, the proposed electron microscopy core would be a collaborative INBRE/COBRE effort. Key personnel involved in implementing the electron microscopy core include:

  • Martha Bickford, Ph.D., has used transmission electron microscopy for more than 35 years in her own research program and has assisted faculty and students in using electron microscopy throughout the last 22 years at the University of Louisville. She will oversee a technician at the University of Louisville who will maintain the Hitachi electron microscope and train users. Also at the University of Louisville, Sanjay Srivastava, Ph.D., FAHA, director of the Pathology and Bio-Analytics Core of the Center for Excellence in Diabetes and Obesity Research, would serve as the associate director of the KBRIN electron microscopy core. Personnel under the direction of Srivastava will prepare tissue for users on a fee-for-sample basis.
  • John Andersland, Ph.D., would also serve as an associate director of the KBRIN electron microscopy core. Andersland is the director of Western Kentucky University’s Ogden College Electron Microscopy Center and is active in student training. He teaches an undergraduate electron microscopy methods course, as well as the electron microscopy portion of a class associated with the Small Genome Project in which students isolate bacteriophages, view their ultrastructure, then sequence their genome. This class has a demonstrable success rate in engaging undergraduate students and piquing their interest in biomedical research, and, as part of the electron microscopy core, WKU’s teaching mission will continue to provide support for sample preparation. At the same time, KBRIN will provide support to assure continued maintenance of WKU’s electron microscopes, as well as provide access for student and faculty research projects.
  • John Balk, Ph.D., PE, would be an associate director of the electron microscopy core. Under his direction, personnel will assist and train users in the characterization of biological samples. Balk is the director of the University of Kentucky Electron Microscopy Center and has 23 years of experience with a variety of electron microscopy techniques, including more than a decade of research group activities at UK. He has repeatedly taught UK’s Materials Characterization course and, in the fall 2018 semester, will offer a new course on Advanced Characterization, the focus of which will be electron microscopy techniques for the analysis of a wide range of materials and samples. Within this structure, sample preparation services will be offered through UK’s College of Arts and Sciences Imaging Center on a fee-for-service basis. Balk will be the point of contact, but would send sample preparation inquiries to Doug Harrison, Ph.D., director of the Imaging Center.

While electron microscopy core work is expected (and encouraged) to happen throughout the state of Kentucky, WKU’s Electron Microscopy Center will serve as a key facility because it hosts the only university-based transmission electron microscope outside of Lexington or Louisville.

“That’s a significant differentiator for WKU,” Andersland said.

WKU’s ultramicrotome, the machine that slices plastic-embedded samples into ultra-thin sections using glass or diamond knives.

What’s ahead: the potential of the KBRIN electron microscopy core

To help reverse a steady decline in transmission electron microscope use, the proposed electron microscope core will encourage the use of electron microscopy across the state in a multi-faceted approach guided by successful practices of previous KBRIN cores.

For example, the electron microscopy core will include an annual electron microscopy workshop, which would unite personnel from the electron microscope facilities at the three partner universities to provide didactic and hands-on classes to train faculty and their students in the theory and practice of electron microscopy, as well as the interpretation of electron microscopic images.

“The workshop would also include scientific talks from established and new microscopists,” Andersland said. “And the location of the workshop would rotate between institutions in order to recruit as many new users as possible. All participants will be invited to join an electron microscopy user group that will provide a forum to discuss research, form collaborations and solve technical issues.”

Additional components of the electron microscopy core include:

  • Funding small pilot project grants to help investigators and their students pay for sample preparation, training and/or beam time fees at any of the three KBRIN network electron microscopy facilities. The grants will enhance both research expertise and infrastructure across the state, help investigators and student trainees add electron microscopy to their repertoire of research techniques and contribute to the maintenance of the three electron microscopy facilities across the state.
  • Requiring student involvement, which will further promote electron microscopy outreach and training. For example, workshop attendance will be restricted to students, post-doctorate students or faculty attending with their students or post-docs. Similarly, pilot project grant applications will require a description of student/post-doctorate involvement in the project. Applications that outline solid training opportunities will be given the highest funding priority as determined by the core directors. Grant recipients or their student trainees will be encouraged to present scientific talks at subsequent electron microscopy workshops, which would promote the continued development of expertise in electron microscopy and potentially generate collaboration across the larger KBRIN network.
  • Showcasing KBRIN electron microscopy core research in articles posted on the core’s website. The goal of this information sharing is to encourage new users by providing technical information and potential contacts for collaboration. Those interested in electron microscopy can also sign up for the KBRIN core’s e-mail list for facility news, communication and networking with fellow investigators and opportunities for interdisciplinary collaborative projects.

WKU—and the Western Kentucky Research Foundation—are no strangers to the power of research collaboration, and the proposed electron microscopy core is no exception. In Andersland’s view, facilitating increased collaboration is among the best outcomes that could result from the KBRIN electron microscopy core.

“When anyone within the KBRIN network has need for biomedical electron microscopy, they can get their work done,” he said. “And by bringing people together, I think it’s more likely that new techniques will be discussed and implemented. That’s certainly my hope.”

Sidebar:

FAQs: An Electron Microscopy Primer

What is light?

Light is made up of photons. These aren’t particles with mass, but can be thought of as packets of energy. Without mass, light is weird; it moves very fast (at the “speed of light”) and can pass through solid objects (like glass). Light can also be created (as from an incandescent light bulb) or destroyed (as when absorbed by black paint).

Light can also behave like a wave. When the amount of energy in a packet changes, its speed doesn’t change; instead, its wavelength changes. Human eyes can only detect light within a specific range of energies, with wavelengths from 400-700 nanometers. “Invisible” light with shorter wavelengths include ultraviolet, X-ray and gamma ray photons. Longer wavelengths include infrared, microwave and radio wave photons.

What are electrons?

Electrons make up atoms along with protons and neutrons. Electrons are negatively charged and balance the charge of protons to give neutral atoms and molecules. Unlike photons, electrons have a mass, although it is very small. Having mass, electrons have a tough time passing through objects without colliding into other electrons, protons and neutrons. Their speed is also variable. But like photons, electrons have wave characteristics (lookup de Broglie wavelength) that depend on the electron’s speed. The faster an electron is moving, the shorter its wavelength; an electron moving at half the speed of light has a wavelength of just 0.004 nm!

The ultramicrotome in action. As the plastic sample (trimmed into a trapezoid shape) moves down across the knife edge it cuts off a slice 100 nm thick.

Why use electrons in a microscope?

Whether you have a beam of electrons or a beam of light, your sample has to perturb that beam for you to see the sample. It turns out that an object has to be roughly half a wavelength wide to perturb the beam. To picture this, imagine a metal stake at the edge of a lake: when the water is calm, the stake blocks small ripples because its width is more than half a wavelength wide. But larger waves crash around the stake as though it wasn’t there. Thus objects smaller than around 200 nm (half of 400 nm) are invisible in a standard light microscope using violet light.  And whoever first calculated the wavelength of an electron probably drooled at the thought of creating an electron microscope with the possibility that it could detect objects as small as 0.002 nm!

How do you use electrons in a microscope?

Using electrons in a microscope is problematic; having mass, electrons can’t make it through air, let alone through a glass lens (which wouldn’t focus it anyway). So everything within the microscope is under vacuum and electrostatic and electromagnetic lenses are used to focus the electrons onto your sample.

In a “transmission electron microscope” or TEM, the electrons must pass through the sample to create an image. Given that electrons have mass, this means samples must be ultra-thin—on the order of 100 nm thick in most biomedical TEMs. Since you can’t see electrons, the electrons are projected onto a plate coated with chemicals that give off visible light when hit by electrons. Thus the plate is bright where electrons have passed through your sample, but dark where they have been scattered.

How do you prepare a biomedical sample for the TEM?

Samples are first preserved with chemical cross-linkers that link molecules to adjacent molecules; think of them as molecular duct tape! Then water is replaced with an organic solvent, which in turn is replaced by a plastic resin. After the samples are placed in small molds, the resin is polymerized by curing in an oven.  The samples are then hard enough to slice 100 nm thick using knives made from broken glass or diamonds and the slices are mounted on 3 mm diameter pieces of copper foil screening. After staining with heavy metals, the samples (where they are lying over a hole) can be viewed in the TEM.

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