Bringing electron microscopy to life
Image: Sharon Frase; researcher and director of electron microscopy at the Cell and Tissue Imaging Center, St Jude Children's Hospital.
In 2009, breakthrough electron microscopy from US-based researchers would change the face of treatments to restore sight to sufferers of retinal degeneration.
For nearly a decade, researchers had thought that the layer of ciliary epithelial cells lining the inside of the eye contained retinal stem cells. Past research had shown that when grown in culture dishes, these cells formed tiny spheres comprising around a thousand cells.
What's more, these spheres could also be cultured to produce more spheres, akin to the self-replication of stem cells. And importantly, these cultured sphere cells also showed gene activation that was characteristic of adult eye cells.
However, research from Dr Michael Dyer from the Department of Developmental Neurobiology at St Jude Children's Research Hospital and colleagues proved otherwise.
Painstaking 3D electron microscopy analysis of every single cell within a sphere indicated that each was pigmented - not typical of stem cells - and also had features of ciliary epithelial cells. At the same time, a comparison of the structures of sphere-forming cells and stem cells revealed fundamental differences.
The impact on retinal degeneration research was profound as scientists worldwide switched from studies of ciliary epithelial cells to embryonic stem cells and induced pluripotential stem cells to treat the disease.
And for Sharon Frase, fellow researcher and director of electron microscopy at the Cell and Tissue Imaging Center at St Jude Children's, the result meant everything.
Frase has already realised that 3D electron microscopy could transform research at the hospital, and the retinal degeneration project was one of several collaborations that would help her to prove it.
As she highlights: "We'd tried serial sectioning to show that every cell in that ciliary epithelial sphere had a pigment granule, but just couldn't define where one cell stopped and the other started."
At the time, Frase had approached FEI, asking researchers if they could take a look at an entire 70 micron sphere, using the Helios focused ion beam SEM.
"Together we did it and the fact that this 3D electron microscopy worked was a real eye-opener," she says. "It answered questions that I don't think we would have been able to prove otherwise."
Neurons within mouse retina have been genetically altered to express placental alkaline phosphatase; a detailed 3D reconstruction of complex synaptic rearrangements is shown [Grahame J Kidd et al, St Jude Children’s].
Come 2010, Frase had also joined forces with Neuroscientist, Grahame Kidd, from Lerner Research Institute, Cleveland Clinic Foundation, and Materials Scientist, Amir Avishai, Case Western Reserve University, Cleveland.
Together the researchers wanted to reconstruct neural pathways within cells, using 3D serial blockface SEM, and devised the best way to label the neurons with placental alkaline phosphatase staining alongside heavy metal counterstaining.
As Frase points out: "These researchers gave seminars on 3D electron microscopy here at St Jude. Avishai is from Materials Science and Kidd is from Biology so we learned a lot from this cross-disciplinary collaboration that you just wouldn't learn otherwise."
"We're still collaborating and I pull a lot from Materials Science," she adds.
At the same time, Frase also teamed up with Douglas Green from Immunology at St Jude Children's to investigate signaling pathways that lead to programmed cell death. Here, for example, the researchers used 3D electron microscopy to characterise structural changes in cells undergoing programmed cell death.
3D SEM reconstruction of portions of two mouse fibroblasts, showing the response of a cell to a drug that causes rupture of the plasma membrane, leading to cell death. Arrows point to intact areas of the plasma membrane flanking the site of rupture [Douglas Green et al, St Jude].
"It's been so essential to have these collaborations over the last eight years, and several are only coming to fruition right now," she says. "During this whole time, I sent samples back and forth to FEI and ZEISS for analysis, I gave presentations to show what we can do with 3D-EM, and I was convincing investigators that this is something we needed at St Jude."
Finally, in the September of 2014, St Jude Children's Research Hospital acquired a FIB-SEM, the Helios 660 DualBeam. As Frase highlights: "It was a hard battle, but I believe that [this instrument] is changing the way we think of electron microscopy and what kinds of questions we can answer. It's going to help people understand biology better."
Indeed, since its installation, research using the FIB-SEM has continued apace. Only last Summer, Michael Dyer, alongside Frase and colleagues, published results from their latest retinal degeneration research.
Having pioneered a method to grow stem cells from neurons, the researchers have shown that stem cells derived from rod photoreceptor cells produce more retinal cells than fibroblast stem cells generated from skin.
The results highlight the importance of the stem cell source in developing retinal degeneration treatments, but as Frase highlights: "I don't know how many samples we analysed using TEM, but we had to use 3D electron microscopy to characterise the structure of the retinal cells." she says.
"We used [our instrument] to find a rod photoreceptor and see its junction with another neuron, the ribbon synapse and other detail," she adds. "3D electron microscopy gives you the hope that you can look at a whole neuron and then zoom into something specific like a synaptic junction. And it has been so important to show how our developmental neurobiology department benefits from this."
Still, practicalities exist. According to Frase, processing 3D electron microscopy data is a massive bottleneck right now that needs to be managed if research is to continue moving forwards. She and colleagues are currently collaborating with computer scientists to find ways to automate analysis but as she emphasises, the problem remains.
"This is a huge huge issue and researchers all over the world are working on this," she says. "The storage and transfer of massive images has to be dealt with and all research is different; if you can threshold [a feature] in the brain, this doesn't mean you can do the same with the mitochondria in a fruit fly model."
However, looking to the future, Frase now believes that correlative microscopy is going to grow in importance at St Jude Children's. As well as the Helios 660 DualBeam, the Cell and Tissue Imaging Electron Microscopy Facility is home to a Tenos VS SEM with in-chamber microtome for in-situ sectioning, and a Tecnai G² F20-TWIN TEM with FEG.
Meanwhile, the Cell and Tissue Imaging Center also houses a Light Microscopy Facility that includes confocal laser scanning microscopy, with a Zeiss LSM 780 and Nikon C-series confocals, as well as Marianas spinning disk confocal imaging systems. Lightsheet microscopy via a Zeiss Lightsheet Z.1 is also available, as is an Elyra PS.1 platform that integrates PALM, STORM and SIM super-resolution microscopies.
"I think between light microscopy, lightsheet technology and more, we're going to be able to combine these systems," highlights Frase. "Correlative microscopy is key here, and to be able to take research from the light to electron microscopy is so important."
"And this is where the microtome becomes really important as we can now sample a large section and study this in its entirety," she adds.
"Research is geared towards allowing translational research to evolve to the patient quickly and collaboration is key." Sharon Frase
But, as with all researchers involved with 3D electron microscopy, limited sample throughput is an issue. Given this, Frase says the Center team works very hard to ensure light microscopy analysis takes place before electron microscopy, with projects flowing back and forth to ensure researchers have all avenues open.
The electron microscopist is also very careful to educate researchers and let them know exactly how long sample preparation and analysis can take using 3D microscopy.
"A researcher may need to use the vibratome to slice a specific area of the brain or look at a basal body in a primary cilia; these tasks take a lot of time," she says. "So we have these honest conversations from the outset and then let the investigator decide, and often they will say, 'it's not necessary'."
And, for Frase, decisions such as this are critical to life in her laboratory at St Jude Children's Research Hospital.
"Research here is geared towards answering the questions that will make the breakthroughs," she says. "My goal is to get 3D electron microscopy functioning efficiently so our researchers can see something they might not be able to see any other way," she adds. "And if by doing so our researchers can answers these questions more quickly, I have accomplished my task."
Backgrounder: What happens at St Jude Children's Hospital
St. Jude Children's Research Hospital, founded in 1962 and located in Memphis Tennessee, is a non-profit pediatric treatment and research facility focused on children's diseases.
The hospital’s mission is to advance cures and means of prevention for pediatric catastrophic diseases through research and treatment and currently has more than 4000 staff, including over 160 researchers. These scientists are engaged in a broad spectrum of research, including discovery-oriented basic science research, the investigation of disease pathogenesis and drug resistance, biobehavioural and quality-of-life research, and therapeutic trials.
As Sharon Frase, Director of Electron Microscopy at the Hospitals Cell and Tissue Imaging Center points out: "I deal with researchers from fourteen disciplines, from cell molecular biology, developmental neural biology and haematology to oncology, veterinary pathology and genetics."
"Research is geared towards allowing translational research to evolve to the patient quickly and collaboration is key," she adds.