Probes, optics for super-resolution microscopy and life in a creative country.
Quantum mechanics fascinated her as a high school student so Ilaria Testa sought out theoretical physicists. “I went to the university to chat with them,” she says. Testa is now a faculty member in applied physics at KTH Royal Institute of Technology in Stockholm and also part of SciLifeLab, which joins researchers at four universities in Stockholm and Uppsala in a creative, scientific way, she says. On the same floor as her lab, teams use various types of imaging or they simulate molecular dynamics computationally; one floor above hers, labs are sequencing and analyzing genomes. “It’s really cool,” she says. The environment exposes her to molecular biology’s open questions. “I think it’s where the ideas have to come from,” she says.
As a physics student at the University of Genoa, she felt drawn to studying molecules. For her PhD, she worked on quantitative methods in single-molecule biophysics and studied transitional states in fluorescent proteins. She became a “barrier breaker” in that supportive faculty opened the door for her from physics to biophysics and optics.
Testa spent part of her doctoral studies and her full postdoctoral fellowship at the Max Planck Institute for Biophysical Chemistry with Nobel laureate Stefan Hell. “I had such an amazing six years,” she says of her time there. “I feel very lucky.” There, she learned “how to think big” and pursue ambitious projects. She worked with a variety of imaging techniques: stimulated emission depletion (STED), reversible saturable optical linear fluorescence transitions nanoscopy (RESOLFT), photo-activated localization microscopy (PALM), and stochastic optical reconstruction microscopy (STORM).
“I feel very lucky.”
She began exploring how to make reversibly photoswitchable fluorescent proteins (rsFPs) compatible with live-cell imaging.
Testa and her team, including paper first author Francesca Pennacchietti, and colleagues at institutions in Russia and the US now present these probes, based on FusionRed, and a custom-built MoNaLISA microscopy platform, which stands for molecular nanoscale live imaging with sectioning ability. 1
Super-resolution is a balance between spatial and temporal resolution and photo-damage, says Testa. For example, STORM can be too slow and STED can be too hard on live cells. The new work balances these factors: she combined ways to optimize probes and the optics to build a system with which labs can look at “small, fast-developing things in living systems.”
Testa chose FusionRed because it is bright and it works well with protein fusions. The new rsFPs show good, fast red-switching behavior. “The more they switch, the better is your spatial resolution,” she says. The probes can be switched between states with green and orange light instead of the UV light needed with previous rsFPs and that is harsh on cells. The MoNaLISA system has a large field of view and it required illuminating in a special pattern: Testa developed a micro-lens configuration to deliver nearly 4,000 foci for simultaneous illumination. “You can see the entire cell, 50 by 50 microns, and it can be increased to 100 by 100,” she says. In her view, a multi-foci light-pattern approach was a missing aspect for RESOLFT-based optical nanoscopy with live cells.
Testa hopes to use the system to study neurobiological questions such as the role of organelles in neurons and the synapse. As part of this she plans to follow proteins in live cells and see how she might multiplex the system she has built with additional colors.
In her growing lab, most but not all members have a physics background. She enjoys discussions with them and other colleagues, which happen especially at Fika, which is the Swedish break for coffee and cake. “They say this is a style of life or a way of thinking,” says Testa. “They really love Fika here.” Sweden tends to the well-being of its inhabitants, the larger questions in society and how each person can contribute to those, she says, “It’s a very creative country, I think.”
Ideas for the lab crop up during Fika or after going for a run. “After sports is a very good moment,” she says, when you look at things differently, which can also happen at the table when sharing a glass of wine with friends and colleagues. Her undergraduate and graduate advisor Alberto Diaspro, who directs nanophysics at Istituto Italiano di Tecnologia, calls Testa a wonderful, 360-degree scientist. “Her eyes, always ready to capture any detail, her brain processing what was going on with enthusiasm,” he says. He and she resonated because they both experience the joy of any new discovery. She was motivated by life and her character to transform her knowledge and background into something that could benefit humankind, he says. In his lab, he cultivated a family sense of belonging, which helps to maintain enthusiasm and respect in good times and in bad. “You are a lucky person if you work in Ilaria’s lab as I was having her in my lab,” he says.
When Testa can find the time, she pursues photography. Although she enjoys reproducing reality, “I try to get away from that, even try sometimes to get away from color,” she says. Black and white photography offers freedom to explore perspective, even a more surrealistic perspective of reality. “I like to use it to catch a moment like I perceive this moment,” she says. Photography shares aspects with microscopy in that it is a “search for the unpredictable,” she says. Experiments are carefully planned but can be unpredictable. Photography, too, “can create something unpredictable.”
Pennacchietti, F. et al. Fast reversibly photoswitching red fluorescent proteins for live cell RESOLFT nanoscopy Nat. Methods https://doi.org/10.1038/s41592-018-0052-9 (2018).
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Conservation Science and Practice (2019)