受激发射损耗显微术(STED)的机理及进展研究

由于光学元件的衍射效应,常规光学显微术的分辨率被限制在半波长左右,无法满足对于亚百纳米尺度的样品进行探测的需求。受激发射损耗显微术(STED)通过引入一束损耗光以受激发射的方式减小有效荧光的发光面积,可以实现超衍射极限的空间分辨率。自提出以来,STED显微术经过了多方面的改进和发展,已被成功地应用于生物医学、材料学等领域,对样品进行多功能超分辨成像。本文详细阐述了STED的机理及其中的关键技术,综述了STED的发展历程及最新进展,并介绍了其具体应用。     

Dissecting tripartite synapses with STED microscopy

The concept of the tripartite synapse reflects the important role that astrocytic processes are thought to play in the function and regulation of neuronal synapses in the mammalian nervous system. However, many basic aspects regarding the dynamic interplay between pre- and postsynaptic neuronal structures and their astrocytic partners remain to be explored. A major experimental hurdle has been the small physical size of the relevant glial and synaptic structures, leaving them largely out of reach for conventional light microscopic approaches such as confocal and two-photon microscopy. Hence, most of what we know about the organization of the tripartite synapse is based on electron microscopy, which does not lend itself to investigating dynamic events and which cannot be carried out in parallel with functional assays. The development and application of superresolution microscopy for neuron–glia research is opening up exciting experimental opportunities in this regard. In this paper, we provide a basic explanation of the theory and operation of stimulated emission depletion (STED) microscopy, outlining the potential of this recent superresolution imaging modality for advancing our understanding of the morpho-functional interactions between astrocytes and neurons that regulate synaptic physiology.     

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer''s attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.