The majority of studies of the living cell rely on capturing images using fluorescence microscopy. Unfortunately, for centuries, diffraction of light was limiting the spatial resolution in the optical microscope: structural and molecular details much finer than about half the wavelength of visible light (~200 nm) could not be visualized, imposing significant limitations on this otherwise so promising method. The surpassing of this resolution limit in far-field microscopy is currently one of the most momentous developments for studying the living cell, as the move from microscopy to super-resolution microscopy or ‘nanoscopy’ offers opportunities to study problems in biophysical and biomedical research at a new level of detail. This review describes the principles and modalities of present fluorescence nanoscopes, as well as their potential for biophysical and cellular experiments. All the existing nanoscopy variants separate neighboring features by transiently preparing their fluorescent molecules in states of different emission characteristics in order to make the features discernible. Usually these are fluorescent ‘on’ and ‘off’ states causing the adjacent molecules to emit sequentially in time. Each of the variants can in principle reach molecular spatial resolution and has its own advantages and disadvantages. Some require specific transitions and states that can be found only in certain fluorophore subfamilies, such as photoswitchable fluorophores, while other variants can be realized with standard fluorescent labels. Similar to conventional far-field microscopy, nanoscopy can be utilized for dynamical, multi-color and three-dimensional imaging of fixed and live cells, tissues or organisms. Lens-based fluorescence nanoscopy is poised for a high impact on future developments in the life sciences, with the potential to help solve long-standing quests in different areas of scientific research.

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基于透镜的荧光纳米技术

大多数活细胞研究依赖于使用荧光显微镜捕获图像。不幸的是，几个世纪以来，光的衍射限制了光学显微镜中的空间分辨率：结构和分子的细节比可见光的大约一半波长（~200 nm）更精细，无法可视化，这对原本很有希望的这一点施加了很大的限制方法。远场显微镜中分辨率的突破是目前研究活细胞最重要的发展之一，因为从显微镜到超分辨率显微镜或“纳米显微镜”的转变为研究生物物理和生物医学研究中的问题提供了机会。一个新的细节水平。这篇综述描述了目前荧光纳米镜的原理和模式，以及它们在生物物理和细胞实验方面的潜力。所有现有的纳米显微镜变体都通过将其荧光分子瞬时制备成具有不同发射特性的状态来分离相邻特征，以使特征可辨别。通常这些是荧光“开”和“关”状态，导致相邻分子及时按顺序发射。每种变体原则上都可以达到分子空间分辨率，并各有优缺点。有些需要特定的转换和状态，这些转换和状态只能在某些荧光团亚族中找到，例如光可切换荧光团，而其他变体可以使用标准荧光标记来实现。与传统的远场显微镜类似，纳米显微镜可用于动态、固定和活细胞、组织或生物体的多色和三维成像。基于透镜的荧光纳米技术有望对生命科学的未来发展产生重大影响，并有可能帮助解决不同科学研究领域的长期探索。