New super-resolution microscopy imaging technology: rapid imaging of neurons

The research group led by Wang Kai from the Center for Excellence in Brain Science and Intelligent Technology of the Chinese Academy of Sciences has published a research paper titled "Super solution imaging of fast morphological dynamics of neurons in eating animals" online in Nature Methods. The team has developed a new type of super-resolution microscopy imaging technology, which solves the two major technical problems of background noise interference and motion artifacts. It can perform super-resolution optical imaging and analysis of the rapid dynamics of neurons in awake animal brains, providing a new tool for exploring the basis of neuronal burst plasticity in animal learning processes.

In recent years, the newly developed super-resolution optical microscopy imaging technology can break through the optical diffraction limit. At present, due to multiple technical difficulties, super-resolution fluorescence microscopy is mostly used for ex vivo cell and brain slice research, and there is no technology that can super-resolution analyze the structure and function of synapses in normal physiological and behavioral states in awake animals. Therefore, the development of new super-resolution optical imaging technologies applicable to conscious animals is at the forefront of neuroscience and optical imaging technology.

The team proposed the Multi Mode Reuse Structured Light Illumination Super Resolution Microscopy Imaging Technology (MLS-SIM). The key innovation of MLS-SIM is the proposal to quickly switch between different line illumination modes in a single line scan imaging process to obtain super-resolution information in three directions, and to propose a new theoretical framework for super-resolution reconstruction to achieve accurate and efficient super-resolution image reconstruction. Under linear fluorescence excitation mode, MLS-SIM can perform continuous imaging of the micro dynamics of dendritic spines and axonal terminals in the cortex of conscious mice with a lateral resolution of 150 nanometers for thousands of frames, and the speed can reach several frames per second; Can tolerate sample motion of 50 microns per second without affecting its super-resolution imaging performance. Furthermore, nonlinear fluorescence excitation can be achieved through picosecond pulse laser, and nonlinear MLS-SIM can increase the lateral resolution to about 100 nanometers while maintaining the same tolerance for sample motion.

MLS-SIM fills the gap in imaging of awake animals using super-resolution microscopy and addresses the shortcomings of previous techniques in imaging time and photobleaching characteristics of live animals, opening up prospects for in vivo microscopic research. Using this technique, the study validated the rapidly changing spike dynamics on neuronal dendritic spines and axon terminals in the awake mouse brain, and quantified the microscopic rapid dynamic changes in neurons during the awake sleep cycle. At the same time, this technology achieved dual color super-resolution simultaneous imaging and analyzed the relationship between the microstructure of PSD-95 protein aggregates and dendritic spine formation. In the dual color imaging experiment, it was found that there are many dynamic small protrusions in the dendritic backbone. The size of this protruding structure is often smaller than the analytical capability of traditional two-photon imaging technology, and can only be dynamically analyzed through super-resolution imaging. Dynamic observation shows frequent small protrusion formation near PSD-95 clusters on dendritic backbones. Furthermore, through statistical analysis of the dynamics over a period of time, it was found that there is a significant co localization phenomenon between PSD-95 clusters on dendritic backbones and small protrusions on the backbones. This newly discovered small protrusion structure and its co localization with PSD-95 may imply a cellular mechanism for dendritic spine formation, providing new evidence for future synaptic plasticity research.

Application of MLS-SIM in cortical super-resolution imaging of conscious mice

This technology enables long-term and large-scale imaging and analysis of subcellular micro dynamics of neurons and other cells in the physiological state of conscious animals, laying the foundation for the application of super-resolution imaging in the field of neuroscience and providing new tools for neuroscience research.

Source: Opticsky