21 Jun 2023
Fluorescence imaging plays its part in study of interactions between individual cells.
The progress of HD is complex but known to be related to dysfunction among astrocytes, particular glial cells in the brain and spinal cord.
But how this dysfunction actually manifests itself in structural terms and the factors that influence its progression are unclear, and tools for structurally rendering nanometer-scale astrocytic interactions have not to date been available.
A project at the University of Rochester has now developed a new imaging methodology combining microscopy, viral tracing and phenotype-specific tagging to reconstruct HD synapses at the nanometer scale. The work was published in PNAS.
"It is one thing to understand the structure of the synapse from the literature, but it is another to see the precise geometry of interactions between individual cells with your own eyes," said Abdellatif Benraiss from the Center for Translational Neuromedicine (CTN), a collaboration between Rochester and the University of Copenhagen.
"The ability to measure these extremely small environments is a young field, and holds the potential to advance our understanding of a number of neurodegenerative and neuropsychiatric diseases in which synaptic function is disturbed."
One element of the new approach is correlative light electron microscopy (CLEM), a technique which combines fluorescence microscopy with high-resolution electron microscopy (EM). On their own, fluorescence imaging can give functional information but is relatively limited in resolution compared to EM, while EM achieves high resolution without functional information.
Using both techniques together has involved inventive optical designs, as in the MirrorCLEM platform developed by Japan's RIKEN research institute and Hitachi High-Tech, where an electron microscope stage is coordinated to a target position defined by a low magnification light microscopy so as to observe the same field of view.
Fluorescence imaging and infra-red branding
The Rochester approach combined CLEM with scanning EM to directly visualize specific astrocyte-synapse interactions. Bespoke numerical analysis methods were then able to extract quantitatively significant information from nanometer-scale structures in these discrete 3D volumes.
"We used CLEM together with 2-channel fluorescence imaging of fluorescence-tagged, retrograde-traced neurons and astrocytes," said the project in its paper. "This combination of imaging modalities allows the capture of relevant light microscopy data prior to serial EM scans, facilitating retrieval of the desired regions of interest in EM and preserving contextual information of the nanometer-scale datasets."
In trials the new methodology was used to compare the brains of healthy mice to mice carrying the mutant gene that causes HD. After tagging axons, motor neurons and astrocytes in a mouse brain, ex vivo examination using multiphoton microscopy was carried out.
As part of the workflow, infrared 2-photon branding created deliberate ablations in the tissue close to the regions of interest, as location markers. Electron microscopy then imaged ultrathin slices of brain tissue to create 3D models of the labeled cells and their interactions.
"The models reveal the geometry and structural relationships between astrocytes and their partnered synapses," commented Carlos Benitez Villanueva from Rochester CTN. "This approach gives us the ability to measure and describe the geometry of the synaptic environment, and to do so as a function of glial disease."