Much more than pretty pictures
By Elise Lamar, PhD
Got a biological question that could be answered by microscopy? The Stowers Microscopy Center can suggest just the right scope for you. Then, after they’ve shown you how to turn it on without destroying a half-million dollar piece of equipment, the team members will marshal their diverse talents to help you acquire, present and make mathematical sense of your startling images. Some may even be suitable for framing.
Tools used to conduct biological experiments, such as microscopes, computers, or DNA sequencing machines, have become mind-bogglingly expensive. So most research centers have “core facilities” in which investigators pool resources to purchase big-ticket items.
But unlike core facilities in many academic institutions, the Stowers Microscopy Center team members, many with advanced degrees in physics or chemistry, often work with investigators to acquire and analyze microscopy data in an extraordinarily collaborative manner. And, if none of the microscopes they have in-house suit your needs, they can custom build one.
A Stowers core value: supporting scientists
Over 100 individuals work in the Institute’s twelve core facilities, which offer expertise in specialties from reptile husbandry to computational biology. The Microscopy Center is among the largest. Their team is on a mission to train their colleagues in all matters relevant to light microscopy, plus keep Stowers’ eighty plus microscopes up and running 24/7. They also supply software for image processing and data analysis, or can even write it themselves if there is a need for something more specialized.
Winfried Wiegraebe, PhD
Winfried Wiegraebe, PhD, leads the Center. He studied microscopy in Germany as a biophysics student at Max Planck Institute for Biochemistry in Martinsried and then completed a postdoc at the Institute for Molecular Biology in Jena. Wiegraebe is a former R&D engineer and product manager at Zeiss, a German company that is to microscopy what Mercedes Benz is to auto manufacturing. At Stowers, Wiegraebe runs the Center assisted by three Group Heads and says his primary purpose is to keep current on new technologies that could be brought to Stowers. In interacting with scientists, he wants the Center staff to go above and beyond. “The members in my group and I are involved in designing experiments and developing new methods,” he says. “In fact, if they want, Stowers labs can outsource complete experiments to us, including experimental design, data acquisition and analysis.”
Case in point: as one part of a study of regeneration, HHMI Investigator at Stowers, Alejandro Sánchez Alvarado, PhD, needed to count dividing cells after a manipulation designed to activate flatworm stem cells, so his group stained thousands of worms with a green fluorescent dye that marks dividing cells. They then asked Sean McKinney, PhD, one of the microscopy Group Heads, to help with analysis.
McKinney imaged a very large number of glowing green dots marking dividing cells in worm tissues using a confocal microscope, an instrument built to reject background fluorescence that might obscure details. He pieced images back together to create pictures of entire worms aglow with fluorescent specks. He and a colleague then wrote a computer program to compare speck number in normal versus experimentally manipulated worms.
McKinney has a doctorate in physics from University of Illinois Urbana-Champaign and completed a postdoc in a protein-engineering lab at the Howard Hughes Medical Institute’s Janelia Farm Research Campus, where he developed novel fluorescent proteins. He calls himself a “hired gun” on the Sánchez Alvarado study, which was published in eLife in 2014 with McKinney as a co-author. To McKinney, this kind of cooperation to solve a problem is business as usual: “We regularly go beyond the routine core operations of maintenance and training.”
Sánchez Alvarado is more expansive: “Sean designed a customized protocol that allowed us to get the most information possible from our data and provided an infrastructure to carry out more complex experiments,” he says. “This is where the Microscopy Center shines brightest. Without them, these efforts would lead to significantly fewer insights.”
Finding problems before the users do
From top: Steve Hoffman, Chris Wood, PhD,
Sean McKinney, PhD
In addition to facilitating cell biology breakthroughs, the Microscopy Center team members spend time doing routine tasks like cleaning intricate components, writing software, and training Stowers scientists on how to use the millions of dollars worth of equipment. Group Heads Chris Wood, PhD, an expert in image processing, and Steve Hoffman, who oversees scope maintenance, supervise many of these tasks.
Wood earned a doctorate in physics in 1998 from University of Missouri-Rolla with a thesis entitled, “Collision dynamics for electron removal from helium and molecular hydrogen by heavy ions,” which he defines as “what happens when two particles smash into each other.” He then spent eight years working as a software developer at consulting companies, where, among other things, he designed computer simulations to assess fluid flow.
Most days, Wood, assisted by Research Specialist Richard Alexander, teaches researchers how to manipulate multiple software packages that quantify imaging data so they can create meaningful plots and tables of the data. This is a non-trivial task, because for some applications, processing an image is more demanding than acquiring it. In some instances, Wood or Alexander might offer to look at the data from an experiment and compile results for the researchers.
Hoffman also brings eclectic training to his job as overseer of equipment. In the 1980s, he earned a business management degree at Kansas State University and then a master’s degree in education at University of Missouri-Columbia, where he specialized in counseling and even worked briefly as a psychologist.
Over the last twenty years, Hoffman has learned to digitize physiological signals, such as brain waves, acquired by medical equipment for computer analysis and become adept as a programmer, first at a contract research institute and then at Cerner Corporation, a supplier of health-care information technology located in the Kansas City metropolitan area. He joined the Microscopy Center in 2007.
Hoffman spends his days “finding problems before the users do” and drives microscope repair when needed. Drive is the operative term, because the center is structured such that the maintenance of instruments, some of which cost nearly a million dollars, is a group endeavor. Each staff member, regardless of job description, is assigned one scope as their baby, an instrument they know inside-out, as part of what Wiegraebe calls “the expert principle.”
“Maintaining and evaluating a system helps us understand how to design better experiments,” says Wiegraebe. “It’s as important for successful science as developing cutting-edge imaging approaches or analysis algorithms.”
To FRET, SPIM, or TIRF?
The Axioplan is capable of acquiring both fluorescence and transmitted light images that utilize various contrast enhancement methods. The motorized filter turret permits multi-channel acquisition.
Most of the Center’s instruments are fluorescence microscopes, meaning that they beam light of a certain wavelength at a specimen, whose components, such as nuclei, membrane proteins, or microtubules, have been stained with dyes that glow when struck by light of that wavelength, making structures visible. Specimens treated with multiple stains resemble collages of glowing red, green and blue patterns revealing the relationship of cellular components. Images are two-dimensional, but confocal microscopes and their cousins called “two-photon” microscopes—often used with very thick specimens—can optically section through a piece of tissue, while a companion computer “stacks” images to create a 3D view.
Beyond that, more specialized techniques are available, depending on whether one needs a flat or 3D image, whether tissue is fixed or living, and (if living) how cells need to keep functioning to answer the question at hand. These applications go by acronyms like TIRF (Total Internal Reflection Fluorescence), FRET (Förster or Fluorescence Resonance Energy Transfer), or SPIM (Selective Plane IlluminationMicroscopy), to name a few. Whatever the technological details, a Center team member knows how to apply it and advise you on whether it’s best for each application.
Take TIRF. One would only apply TIRF to answer a question that required limiting your view to a very thin 2D plane with no confounding signals intruding from above or below that plane. As a graduate student in the lab of Investigator Rong Li, PhD, Sarah Smith, PhD, employed TIRF to analyze how vesicles moved into and out of yeast cell membranes. For one study, Li’s group needed to be 100% sure that two proteins resided in different cellular membranes. By confocal microscopy, the proteins appeared to coalesce into a single blob, but TIRF analysis revealed that the proteins were actually embedded in closely apposed but different membranes.
After earning her doctorate in 2013 from the University of Kansas Medical Center, Smith joined the Microscopy Center as a research specialist. Her expertise in microscopy (as an undergrad she won an award from the National Society of Physics Students for building an instrument called a scanning tunneling microscope) plus her familiarity with Stowers science has given her a leg up in establishing collaborations with multiple Institute scientists.
One collaborator is Associate Investigator Sue Jaspersen, PhD, with whom Smith worked in applying FRET, a completely different application of microscopy. FRET measures the transfer of energy from one fluorescing molecule “excited” by light to a nearby molecule. Thus, biologists doing structural studies might use FRET to figure out whether proteins actually touch each other. In this case, Jaspersen, who studies multi-protein nuclear structures that help organize chromosomes in yeast, worked with Smith to use FRET to determine how these structures insert into yeast nuclear membranes and then reconstruct themselves protein-by-protein when cells divide.
Another fruitful Stowers collaboration occurred recently between the Microscopy Center and Associate Investigator Matt Gibson, PhD. Gibson needed to image embryonic fruit fly cells residing in epithelial sheets to determine if a specific mutation disturbed how cells divide within that sheet. Doing that imaging required a strategy to view components of a living cell’s mitotic spindle (the machinery that drags chromosomes into daughter cells during mitosis) long enough to capture individual cells dividing.
So McKinney, working with Center Laboratory Assistant Amanda Kroesen, scavenged parts from other confocal microscopes to build an in-house SPIM microscope. This is an instrument that allowed them to illuminate tissues from the side, rotate them to view from all sides, and image them for long periods of time without the laser “bleaching” or fading the fluorescent signal—all while quantifying the extent to which the mitotic spindle tilted.
The Gibson Lab published that work, complete with time-lapse films of dividing cells, in Nature in 2013, with McKinney and Kroesen as co-authors. It reveals that alignment of a cell’s mitotic spindle relative to the surface of a cell layer is critical for epithelial integrity and that mutations that cause the spindle to list significantly disrupt an epithelial sheet. The work has implications for cancer: disorderly division of epithelial cells is a hallmark of carcinomas.
For experiments that require imaging of developing cells and organisms, sometimes over the course of several days, the Microscopy Center calls on fellow research specialist Jeffrey Lange, PhD. Lange uses advances in materials science learned during his PhD studies in analytical chemistry at Kansas State University to design and fabricate devices tailor-made to specific live samples ranging from tiny yeast to whole worms. His goal? To keep them under the lens of the microscope while still remaining free to move about. Lange’s devices have enabled Stowers researchers to capture and study the details of processes such as fruit fly ovary development and flatworm locomotion.
Until recently, this kind of robust scientific cross-talk between biologists and people who provide technical support has not been common. But that cultural divide may be dissolving given the enormous technical know-how required to analyze biological data today. Wiegraebe notes with pride that historically physicists have often demonstrated a “let’s-get-the-job-done-as-a-team” spirit, enabling them to rack up significant accomplishments, such as the moon landing. “The management at Stowers knows that people are more productive when they work together,” he says. “So they established the core services to provide investigators with collaborative help from specialists or consultants.”
A new kid on the microscopy block
Although a powerful tool, conventional light microscopy can’t yet hold a candle to electron microscopy (EM) when it comes to resolution. EM (the purview of a different Stowers core) can distinguish features of particles less than a nanometer in size and take snapshots of large protein complexes, like Jaspersen’s centrosomes. But EM sample preparation is onerous and tissue must be fixed (dead): so if you need EM level magnification, you won’t be twirling living tissues around making movies of them.
But stay tuned: new “super-resolution” light microscopes can image structures smaller than 200 nanometers, the limit of conventional light microscopy. Jaspersen recently used one to figure out how a dividing yeast cell knows to make just one copy of its centrosome. She and collaborators from University of Colorado, Boulder, suspected that the nuclear factor Sf1 controlled centrosome duplication. Thus, they mutated, or disabled, Sf1 in yeast and applied a super-resolution technique called SIM (for Structured Illumination Microscopy) to peer into the nuclei of dividing cells and check if they contained the right number of centrosomes, namely two.
Sure enough, many didn’t. Some yeast harboring mutant Sf1 exhibited more than two centrosomes, sometimes separated by less than 100 nanometers. Conventional microscopy would simply have mistaken these two dots for one, just as a double star might appear as a single point through a backyard telescope. Moreover, super-resolution imaging gave the team a way to count centrosomes in large numbers of cells to allow statistical comparison of mutant and control cells. This work tagged Sf1 as a key factor controlling centrosome duplication, which is important, as aberrant centrosome duplication occurs in several diseases, including cancer. Jaspersen and collaborators published the study this summer in PLoS Genetics.
Zulin Yu, PhD, research specialist in the Microscopy Center, was a co-author on that paper. Yu joined the Center in 2010 after earning a biophysics doctorate at the Chinese Academy of Science in Beijing and worked as a postdoc with Stowers Associate Investigator Ron Yu, PhD. Zulin Yu easily made the move from postdoc to core center because he can be an expert on multiple projects and is currently the center’s super-resolution point person. This year, for example, he contributed not only to the Jaspersen study, but was co-author on a paper published in Genetics from the lab of Investigator Scott Hawley, PhD. That study applied SIM technology to show how components of another multi-subunit nuclear complex called the synaptonemal complex were arranged.
“As a postdoc in Ron’s lab I used to stop by the microscopy office and ask Winfried and Sean questions all the time,” says Yu. “They were always glad to help me. Now it’s my job to help many different scientists solve problems that are often quite challenging.”
More beautiful than the real thing
Rong Li, a cell biologist, agrees: “The job of a microscopy core now is not just to help us acquire beautiful images and time-lapse movies but to develop unbiased and reliable methods to quantify signal intensities, fluctuations, and dynamic movement of cells and intracellular structures.”
Although he is a microscopist first and foremost, Wiegraebe is adamant that mathematical analysis is where it’s at: “The days of just pretty images are over!” he says. “Go to any cell biology meeting and you’ll see that the future of microscopy is quantification: this field is no longer descriptive.”
Paradoxically, however, both Li and Wiegraebe relish microscopy’s aesthetic side. Li was delighted when “data” from her lab was chosen for display at “Life: Magnified,” an exhibit this summer at Washington DC’s Dulles Airport. That project featured dazzling microscopy images from labs around the world, among them portraits of tick mouths, lizard toe hairs, liver cells, and (from the R. Li Lab) a single skin cell radiating fluorescent red and green cytoskeletal filaments like a medusa head from a royal blue nucleus. (Related story: Procrastination Pays Off)
Wiegraebe loves contemporary art and has worked as a docent at Kansas City’s Kemper Museum of Contemporary Art for eight years and more recently at Kansas City’s Nelson-Atkins Museum of Art. (Unsurprisingly, the Microscopy Center website has links to pages showing the historic relationship of art to science.) He argues that artists and scientists have more in common than one might think, because both make sense of the world by looking at things carefully. As examples he cites nineteenth century biologists, such as German microscopist Ernst
Haeckel who made delicate sketches of micro-organisms that people still admire, as blurring the line between art and science. “Haeckel was a Romantic scientist and promoter of Charles Darwin who thought that being a good scientist meant being a good artist,” says Wiegraebe. “His drawings were often more beautiful than the real thing, probably because many in the nineteenth century believed that the perfection of the world is reflected in its beauty.”