Researchers at the McKelvey School of Engineering at Washington University in St. Louis have developed a new technique that allows for higher-resolution imaging of small objects like neurons. The technique, called plasmon-enhanced Expansion Microscopy (p-ExM), improves on the existing method of expansion microscopy (ExM). This advancement is described in a recent paper published in the journal Nano Letters.

In conventional microscopes, lenses are used to make objects appear larger and more visible to the human eye. However, ExM works differently—it makes the object itself larger. Scientists use fluorophores, which are tiny light-emitting markers, to coat the sample (such as a cell). Then, the sample is embedded in a gel that expands when it comes into contact with water. As the sample grows larger, the fluorescent labels trace the outlines of small features that are otherwise difficult to see, such as the thin branches (dendrites) of brain cells.

However, expansion microscopy has a drawback—the light signal emitted by conventional fluorophores loses more than 50% of its intensity during the preparation and expansion steps. To overcome this issue, the researchers at Washington University used ultrabright fluorescent markers called plasmonic-fluors (PFs), which were developed for other applications in 2020 by Srikanth Singamaneni, a professor in the Department of Mechanical Engineering & Materials Science.

The PFs are constructed from a core particle of gold wrapped in a silver shell, and they are covered with additional materials, including conventional fluorophores. This design protects the fluorophores from harsh chemicals and enhances the brightness of the light signal emitted by them. The use of PFs enables researchers to map neural networks and study the connections between neurons more effectively.

The gold-silver nanoparticle in the PF acts as an antenna, pulling in more light into the fluorophores. The interaction between the nanoparticle and the fluorophores also causes the fluorophores to emit more photons, resulting in a significantly brighter signal. Compared to conventional fluorophores on their own, PFs are nearly four orders of magnitude brighter. Additionally, the fluorophores in PFs are directly attached to the nanoparticle, preventing signal dilution when the sample expands.

To demonstrate the efficacy of plasmon-enhanced expansion microscopy, the researchers used it to study a sample of neurons from the hippocampus region of the brain. When two neurites (budding branches of neurons) are too close to each other, they cannot be resolved individually. However, after labeling the cells with ultrabright plasmonic-fluors and expanding the sample, the researchers were able to count the number of neurites, measure the total area of the neurites, and determine the length of individual neurites. The expanded sample revealed 2.5 times more neurite terminal points than were visible before expansion.

Comparing the performance of plasmonic-fluors to conventional fluorophores, the researchers found that plasmonic-fluors retained about 76% of the light signal, while the fluorophores retained less than 16%. Importantly, plasmon-enhanced expansion microscopy is compatible with existing ExM protocols, meaning that plasmonic-fluors can be used instead of conventional fluorophores in future studies. Moreover, plasmonic-fluors can be created from any fluorophore that suits the researchers’ requirements.

The development of plasmon-enhanced expansion microscopy opens up new possibilities for high-resolution imaging and mapping of tiny structures, particularly in the field of neuroscience. The brighter Fluorescent Markers Enable scientists to study the intricate details of neurons and their connections, leading to a deeper understanding of the brain and its functions.

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