George Langford

George Langford

Back in the 1980s, I was astounded to be able to see individual microtubules in a squid giant axon the first time I looked through Robert Allen’s video microscope. As a postdoctoral fellow under the mentorship of Shinya Inoue, I observed spindle microtubules in living cells with extraordinary clarity through his polarizing light microscope—they were more exquisite than I could have imagined, having only observed organelles such as mitochondria under a conventional differential interference contrast or phase contrast microscope. Over the past 30+ years, that feeling of amazement has been consistently rekindled as microscope technology progresses at a blistering pace. Just last summer at the Howard Hughes Medical Institute (HHMI) Janelia Research Campus Advanced Imaging Center (AIC), application scientists helped me observe cortical actin filaments at sub-diffraction resolution as they formed finger-like projections on the surfaces of pancreatic beta cells—data that ultimately changed the direction of my lab’s research project. My experience at the AIC was truly incredible—their full-service support from lodging, to cell culture, to not-yet-commercially-available cutting-edge equipment, through image analysis support, made me want to shout praises for the Center from Janelia’s stunning glass rooftops (and the ASCB Newsletter).

“[The AIC] has been a shining example of what American science does very well, which is to share and expand science throughout the world,” says Kile.

At the AIC, my research group had the opportunity to use the original Structured Illumination Microscope (SIM) developed by Mats Gustafsson. Using the Total Internal Reflection Fluorescence (TIRF) mode of SIM to achieve super-resolution visualization of actin filaments, we were able to observe the movement of actin patches in pancreatic beta cells that we subsequently identified as glucose-sensing microvilli. With experts on hand to help with quantitative image processing and motion analysis, we established that the movement pattern changed in response to the glucose concentration. This discovery represented the first time these sub-diffraction limited structures were observed in living beta cells. This set of observations had an extraordinary impact on our research program and led us to focus on the role of the actin cytoskeleton, not only in insulin secretion, but also in glucose sensing by the GLUT2 transporter on beta cell microvilli.

Super resolution multi-color TIRF-SIM (Gustafson) images of actin cytoskeletal dynamics in living cells. (A) Cortical actin cytoskeleton in COS-7 (mRuby-LifeAct); (inset) high mag. Bars, 5 and 1 μm. (B) Cortical F-actin (mEmerald-LifeAct) and clathrin-coated pits (mKOrange-clathrin) in INS-1 832/13 cells; (inset) high mag. Bars, 1 μm and 300 nm. (C) Motile actin-derived membrane projections in INS-1 cells from a time-lapse video sequence. (D) 3D kymographs of the movement. (E) Heat color map of 3D kymograph. Bars, 300 nm.

Super resolution multi-color TIRF-SIM (Gustafson) images of actin cytoskeletal dynamics in living cells. (A) Cortical actin cytoskeleton in COS-7 (mRuby-LifeAct); (inset) high mag. Bars, 5 and 1 μm. (B) Cortical F-actin (mEmerald-LifeAct) and clathrin-coated pits (mKOrange-clathrin) in INS-1 832/13 cells; (inset) high mag. Bars, 1 μm and 300 nm. (C) Motile actin-derived membrane projections in INS-1 cells from a time-lapse video sequence. (D) 3D kymographs of the movement. (E) Heat color map of 3D kymograph. Bars, 300 nm.

Benjamin Kile, professor at Monash University in Melbourne, Australia, also benefited immensely from the AIC by sending his lab members to use the lattice light sheet microscope developed by Eric Betzig, 2014 Nobel laureate. “We had a theory that the DNA was being released from the mitochondria during apoptosis—that was a new idea that didn’t have a lot of support. We wanted to watch mitochondria in real time in live cells, and the lattice light sheet microscope [at the AIC] showed us unequivocally it was happening,” Kile said. Their work with the AIC was recently published. “[The AIC] has been a shining example of what American science does very well, which is to share and expand science throughout the world,” says Kile.

Continuing a Long Tradition

Cell biology has a long tradition of sharing new instrumentation that was championed long ago at the Marine Biological Laboratory (MBL), in Woods Hole, MA. Allen and Inoue and their collaborators developed new video microscopy techniques at the MBL in the 1980s and made their instruments available to others before commercial systems were available. Their eagerness to provide access to other scientists accelerated research and led to major breakthroughs.

The AIC team…provides detailed technical consultation to prospective applicants prior to proposal submission, onsite technical support, and data analysis even after the imaging is over.

Likewise, HHMI, in collaboration with the Gordon and Betty Moore Foundation, created the AIC to make pre-commercial instruments developed at Janelia accessible to life scientists from nonprofit organizations all over the world. “What makes the AIC unique is the degree of the support it provides. It covers the lodging of visiting scientists, the basic experimental costs, 24-hour access to the assigned instruments, and even access to Janelia’s latest technologies such as newly developed imaging probes,” said AIC director Teng-Leong Chew. The AIC team also provides detailed technical consultation to prospective applicants prior to proposal submission, onsite technical support, and data analysis even after the imaging is over. “The AIC staff is fantastic, there’s a technical expert for each of the machines; they’re on-call, and they’re incredibly accommodating,” said Catherine Galbraith, professor at Oregon Health & Science University, who recently used AIC’s iPALM to look at the structure of actin.

The AIC’s involvement in each project starts long before an investigator even sets foot near the peaceful pond by Janelia’s entrance. “We sent the AIC our [stem] cells, which are difficult to grow, in advance. The AIC cell culture specialist successfully grew them even before we arrived, so we didn’t lose any time. While we were there, we used the iPALM microscopy to get the first super-resolved images of pluripotent stem cell adhesion complexes,” said Johanna Ivaska, professor at the University of Turku in Finland.

Chew has worked with Janelia instrument developers to maintain a robust pipeline of cutting-edge equipment, and to provide extensive support to ensure maximum efficiency for scientists while they are in residence at the AIC. “Once the data are collected, AIC data scientists help the investigator analyze the data, which is often the biggest hurdle for interpretation of these advanced microscope systems,” explained Jennifer Lippincott-Schwartz, Janelia group leader and former ASCB President who now serves on the AIC review panel that selects applications from those who want to use the facility.

How to Apply

Anyone can take advantage of the AIC; all it takes is a well-formulated application. “A successful application requires that the project match the capability of the instrument, and that it has strong justification for using the AIC—any project that could be tackled by commercial instruments does not qualify. We also want to make sure that the reagents and the specimens are robust enough, and that they can be safely handled at Janelia. A project needs to be accompanied by solid quantitative approaches. The AIC is the perfect place to generate preliminary data for your brilliant and bold ideas. If your project challenges the paradigm, we especially would love to help, which could give your research a competitive edge,” Chew said.

The AIC is the perfect place to generate preliminary data for your brilliant and bold ideas.

“It’s truly an amazing program; it’s a dream. If I were a graduate student or postdoc, I would absolutely jump at the opportunity to come here….Suddenly you will have a team of experts who are devoted to your project, and have new ideas to inject into your project,” said Lippincott-Schwartz. She recommends, though, that the PI also be involved in the experiments with a grad student or postdoc. “It’s important for the PI to come for at least a day or two, if not longer, to see the whole system and understand the strengths and limitations of the technology, so that it can be integrated into the conceptualization of the project.”

AIC’s comprehensive approach makes the center stand out as a unique facility for accessing emerging imaging technologies, unlike any other in the world. Not only does the AIC offer the opportunity to perform state-of-the-art microscopy to explore paradigm-shifting ideas, it also provides onsite guest suites with sweeping views of Janelia’s tranquil landscape. Plus, to help with the late nights at the AIC’s microscopes: coffee and astounding glimpses at your cells are both free.

Acknowledgments

I thank Christina Szalinski for help with editing the manuscript, Torsten Wollert for help with the figure, and the AIC scientists for support during my visit to the Janelia Research Campus.

George Langford

George M. Langford is Distinguished Professor at Syracuse University.