DHO-enabled 3D dSTORM unlocks insights into nanoscale structure of the brain
Using the SPINDLE module with a double-helix phase mask and dSTORM single molecule localization microscopy, Jason Aoto and colleagues have started to unravel the relationship between nanostructures and function in the brain by mapping the spatial arrangement of key proteins across neural synapses.
Why this is important
The human brain has trillions of connections, called synapses, which are responsible for transmitting chemical and electrical signals from one neuron to the next. Mapping this processing infrastructure is a critical step on the path to developing therapeutic approaches to diseases related to memory and addition, which are associated with synaptic defects.
Enabled by DHO technology, the Aoto Lab at the University of Colorado at Anschutz and collaborators were able to study the nanoscale organization of key trans-synaptic proteins and identify how these molecules coordinate brain signaling.
The science
Above all else, the brain must be able to process information quickly. This means that all pre-synaptic release and post-synaptic receptors must be coordinated in some way. Critical proteins for synaptic connection have been shown to organize in certain regions of high density known as subsynaptic densities (SSDs) and are aligned trans-synaptically to form nanocolumns. While the molecular composition of these nanocolumns is known, the mechanism of SSD formation and trans-synaptic alignment is not well understood. Neurexins are proteins that are suspected to facilitate these operations, as they physically connect synapses and recruit other proteins to the cellular barrier.
For their study, the Aoto Lab focused on two neurexins that localize to the same individual presynaptic neurons but that are associated with different signaling pathways. These proteins were imaged in mouse brain tissue, using two-color direct stochastic optical reconstruction microscopy (dSTORM), allowing the researchers to identify neurexin-3 as a vital component that helps to organize the excitatory synapses (triggering an action potential) in the brain’s hippocampus. They also found that neurexin-1 and neurexin-3 are arranged indistinct, non-overlapping regions, aligning with their respective partners on the other side of the synapse.
How Double Helix Optics made it possible
Synapses are 3D structures associated with 3D chemical and electrical processes that cannot be fully understood with 2D imaging. Specifically, Aoto and colleagues sought to decipher the spatial relationship between two key trans-synaptic proteins, which required two-color localization assays over a depth range of 3 µm. This task was made possible by attaching DHO’s SPINDLE module with twin DH1 phase masks to a Nikon NSTORM super-resolution microscope at CU Boulder’s BioFrontiers Advanced Imaging Core and localizing the raw images using DHO 3DTRAX. Additionally, DHO’s 3D technology allowed the researchers to minimize data analysis artifacts and incorrect conclusions by avoiding the false positive results that are typically encountered in similar studies using 2D microscopy.