单 分 子 定 位 超 分 辨 显 微 技 术 在 神 经 生 物 学 中 的 应 用（特 邀）

Significance Fluorescence microscopy technology is one of the most direct and effective method for studying the structure and function of complex biological systems. Neurobiology involves the study of the structure, function, development, genetics, and pathology of the nervous system, which is essential for understanding how the brain controls behavior, processes information, and changes under disease states. Conventional fluorescence microscopy has become a commonly used tool in neurobiology and is widely applied to the study of neuronal connections and information transmission processes. However, traditional optical microscopes are limited by diffraction, which restricts scientists from observing nanoscale structures. The 2014 Nobel Prize in Chemistry was awarded for super-resolution fluorescence microscopy technology, which has developed into a highly effective research tool for subcellular fluorescence imaging and resolving organelle structures. We introduce the main single-molecule localization super-resolution microscopy techniques, elaborates on the basic principles of three-dimensional single-molecular localization microscopy (3D-SMLM), and describes point spread function (PSF) fitting, aberration analysis, and multi-color imaging techniques. We also summarize the research results of single-molecule localization super-resolution microscopy in the structural analysis of neurons at the nanoscale, including the morphological changes of pathological signals in Alzheimer disease (AD), such as β-amyloid and tau-protein aggregates, and their mechanisms of interaction with neuronal proteins. Finally, we explore the future development prospects of single-molecule localization microscopy (SMLM) technology. Progress First, the basic imaging principles of single-molecule localization technologies, including photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT), are summarized (Fig. 1). The growth of cells occurs in a three-dimensional space, surrounded by other cells, extracellular matrix, and tissues. The refinement of their structure and function requires higher axial resolution in superresolution microscopy technology, leading to the development of 3D-SMLM. The key to 3D-SMLM is to characterize the axial information of a single molecule using the planar shape of the PSF. Major principles based on astigmatism method, double-Helix PSF (DH-PSF), biplane-PSF, 4Pi-PSF and supercritical angle localization microscopy (SALM) are compared (Fig. 2). Given that aberrations caused by instruments and biological samples are often introduced during the imaging process, adaptive optics technology is used to compensate for aberrations through optical equipment to improve imaging quality. Alternatively, an in situ PSF model is directly constructed from the obtained single-molecule dataset to capture the true aberration morphology of biological data and improve positioning accuracy (Fig. 3). The study of biomolecule interactions typically requires imaging multiple target molecules in the same experiment. Multi-color SMLM technology not only provides high-resolution structural information, but also analyzes the spatial structure and interactions of different target molecules (Fig. 4). Moreover, the excellent spatial resolution and potential for specific target recognition of single-molecule localization super-resolution microscopy technology have been demonstrated in neurons and the neurodegenerative disease AD. Key discoveries include the periodic membrane skeleton in neurons (Fig. 5), dynamic changes in the nanostructured domains of synaptic proteins (Fig. 6), and applications in amyloid fibril aggregation and neurofibrillary tangles (Fig. 8). Furthermore, in situ 3D-SMLM enables high-throughput quantitative analysis of high-density neuronal dendritic spine structures in brain tissue slices (Fig. 7). The arrangement of fibrils in the center of β-amyloid plaques is captured and resolved in the axial direction, and local details of individual fibrils are displayed, allowing for the tracing of their growth morphology (Fig. 9). Finally, suggestions for research on single-molecule localization super-resolution microscopy technology in neurobiology are presented. Conclusions and Prospects SMLM has overcome the spatial resolution limitations caused by optical diffraction, achieving super-resolution imaging by accurately locating the position of a single fluorescent molecule. This breakthrough has significantly advanced modern life sciences and biomedical research. Its ability to analyze fine structures at the nanoscale, dynamically map changes in response to pathological and physiological activities, and quantitatively assess high-throughput, high-density structures in three-dimensional in situ environments highlights the broad potential applications of SMLM. In the future, SMLM can be further developed for in situ super-resolution analysis of complex biological samples and exploited as a quantitative biological analysis tool in neurobiology research.