Super-Resolution Microscopy Helps Map Hidden Neuronal Architecture

As a researcher interested in the architecture and organization of neurons within mammalian brains, Christophe Leterrier, a neuroscientist at the French National Centre for Scientific Research-Aix Marseille University, understands the power of peeking into these tissues using super-resolution microscopy. “We use this to try to understand several key structures that are unique to neurons that allow them to have their very unique shapes and functions,” said Leterrier.

Advanced microscopy techniques help Leterrier and his team understand neuronal architecture and function. In addition to this, some of the vivid images acquired by the team make it to the covers of journals and come in handy in communicating science to the public.

Single-Molecule Localization Microscopy Offers Molecule-Scale Resolution

Leterrier and his team isolate cells from the hippocampal region of rat brains and culture them on cover slips in lab dishes for imaging. This method of culturing neurons at a low density enables them to visualize the subcellular distribution of proteins.¹ “It doesn't recapitulate everything that happens in a real brain, but to do subcellular work and understand things at the nanometer scale, [this] is the best model we have,” said Leterrier.

They then subject these cultured cells to advanced microscopy techniques that help visualize the subcellular organization of individual molecules. The diffraction, or bending, of light waves in standard optical microscopy techniques results in blurred images of structures smaller than about 200 nanometers.

Leterrier and his team circumvent this limitation by employing single-molecule localization microscopy (SMLM), a super-resolution microscopy technique that overcomes the diffraction limit of light, helping achieve molecular-scale resolution.²

SMLM methods involve switching fluorophores—bound to specific molecules within the brain tissues—on or off while visualizing consecutive image frames. The signal intensity helps identify the center position of the fluorophores, which enables the localization of specific molecules. Signals collected over thousands of frames superimposed into a single-plane image provide insight into the spatial distribution of molecules.

While this technique offers information at a high resolution, it can only offer snapshots of fixed cells. “Imagine you want to understand soccer or tennis by just looking at fixed photographs,” said Leterrier. “You will need to have tons and tons of photographs to get a feeling of how the game is played,” he explained. “[So], now we are branching off to other techniques.”

Structured Illumination Microscopy Enables Super-Resolution Live-Cell Imaging

One of these techniques is structured illumination microscopy (SIM), a high-speed super-resolution fluorescence microscopy technique.³ The method involves using patterned, structured light to illuminate samples, wherein analyzing the patterns created by the interaction of the light and sample gives information about the dynamics of molecules within the sample.

“You can do live-cell imaging [with SIM], but you don't get as [good] a resolution,” remarked Leterrier. While SMLM can resolve points as close as 20 to 50 nanometers, SIM offers resolutions of only about 100 nanometers.

Despite their differences, Leterrier cannot pick a favorite between the two microscopy methods. “The expertise in the lab is primarily SMLM, and we've done a lot of exciting work with it. So, it’s been always dear to my heart,” he said. But now with the aim of investigating molecular and cellular dynamics, the team is increasingly turning to SIM. “So, I would say the old love is SMLM. The new love is SIM, but I love both of them.”

Leterrier believes that combining the two methods can give researchers powerful information. Using cultured cells for SIM and then fixing them and subjecting them to SMLM could offer a fuller picture of the live-cell dynamics.

Understanding structural dynamics of how different cellular structures assemble, are modulated, and transformed during pathological events like neurodegeneration can offer deeper insight into their roles in the brain. “We can infer and make hypotheses about the function looking at fixed photographs,” said Leterrier. “But now that we know how things move, we can make much better hypotheses and then test them in a functional way.”

Microscopy for Science Communication

While the researchers use microscopy to answer biological questions, “I am always very attentive to the aesthetic aspect of things because I'm doing this in part because I think these neurons are beautiful,” said Leterrier.

He often finds himself going back to these images, tweaking and processing them. “Basically, [you can] have some fun with your data to make a nice image,” he said. While these modified images cannot be used to quantify results, they can give rise to vivid and powerful illustrations.

“It helps communicate the science and make images that are then seen by a lot of people,” said Leterrier.