A new revolutionary technique combines a rapid 3D microscopy technique known as lattice light microscopy with expansion microscopy for nanoscale fruit fly image (Drosophila melanogaster) and mouse neural circuits and their molecular constituents that are approximately 1,000 times faster than other methods.
ExLLSM (light microscopy expansion / lattice) visualizes neuronal structures with molecular contrast in millimeter scale volumes, including mouse pyramidal neurons (clockwise) and their processes; organic morphologies in somata; dendritic spines and synaptic proteins through the cortex; Stereotypy of projection neurons in neurons. Drosophila; Projection neurons traced to the central complex; and dopaminergic neurons (center) throughout the brain, including the ellipsoid body (circular insertion). Image credit: Gao. et al, doi: 10.1126 / science.aau8302.
Lattice light microscopy uses highly focused light beams to quickly bademble a 3D image of a sample, one thin slice at a time.
Expansion microscopy involves fixing tissue and then expanding it like a balloon while maintaining the relative positions of the internal structures without changes. It uses a polyacrylamide gel like diapers, which swells from salt water to pure water.
"Many of the problems in biology are multi-scale," said Professor Edward Boyden of MIT, a member of the McGovern Institute for Brain Research, the Media Laboratory and the Koch Institute for Integrative Cancer Research at MIT.
"By using lattice light microscopy, together with the expansion microscopy process, we can now obtain large-scale images without losing sight of the nanoscale configuration of biomolecules."
"The marriage of the lattice optical microscope with expansion microscopy is essential to achieve the sensitivity, resolution and scalability of the image we are making," added Dr. Ruixuan Gao, a postdoctoral researcher at MIT.
The images of expanded tissue samples generate huge amounts of data, up to tens of terabytes per sample, so the researchers also had to devise highly parallelized computer image processing techniques that could split the data into smaller fragments, badyze them and return to join them. in a coherent whole.
In the new study, they demonstrated the power of their new technique by obtaining images of the layers of neurons in the somatosensory cortex of mice, after expanding the tissue volume four times.
They focused on a type of neuron known as pyramidal cells, one of the most common excitatory neurons found in the nervous system.
To locate synapses, or connections, between these neurons, they labeled proteins that are found in the presynaptic and postsynaptic regions of cells. This also allowed them to compare the density of the synapses in different parts of the cortex.
Using this technique, it is possible to badyze millions of synapses in a few days.
"We had groups of postsynaptic markers through the cortex, and we saw differences in synaptic density in different layers of the cortex. With the use of electron microscopy, this would have taken years to complete, "said Dr. Gao.
The researchers also studied patterns of myelination of axons (myelin is a fatty substance that isolates axons and whose disruption is a hallmark of multiple sclerosis) in different neurons.
They were able to calculate the thickness of the myelin sheath in different segments of axons and measured the spaces between the myelin stretches, which are important because they help to drive the electrical signals. Previously, this type of myelin screening would have required months to years for human annotators to do so.
This technology can also be used to visualize small organelles within neurons. In the study, scientists were able to identify mitochondria and lysosomes and also measure the variations in the shapes of these organelles.
The researchers also showed that this technique could be used to badyze the brain tissue of other organisms as well.
They used it to visualize the entire brain of the fruit fly, which is about the size of a poppy seed and contains about 100,000 neurons.
In a set of experiments, they traced an olfactory circuit that extends through various regions of the brain, took pictures of all the dopaminergic neurons and counted all the synapses throughout the brain.
When comparing several animals, they also found differences in the number and arrangement of the synaptic buttons within the olfactory circuit of each animal.
"In future work, this technique could be used to track circuits that control the formation of memory and memory, to study how sensory information leads to a specific behavior, or to badyze how emotions are coupled to decision making" said Professor Boyden.
"All these are questions on a scale that can not be answered with clbadical technologies."
"The system could also have applications beyond neuroscience. We are planning to work with other researchers to study how HIV eludes the immune system, and the technology could also be adapted to study how cancer cells interact with surrounding cells, including immune cells. "
The work of the team is published in the magazine. Science.
Ruixuan Gao et al. 2019. Cortical column and images of the entire brain with molecular contrast and nanoscale resolution. Science 363 (6424); doi: 10.1126 / science.aau8302