Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002, USA.
Annu Rev Cell Dev Biol. 2010;26:285-314. doi: 10.1146/annurev-cellbio-100109-104048.
Fluorescence imaging methods that push or break the diffraction limit of resolution (approximately 200 nm) have grown explosively. These super-resolution nanoscopy techniques include: stimulated emission depletion (STED), Pointillism microscopy [(fluorescence) photoactivation localization microscopy/stochastic optical reconstruction microscopy, or (F)PALM/STORM], structured illumination, total internal reflection fluorescence microscopy (TIRFM), and those that combine multiple modalities. Each affords unique strengths in lateral and axial resolution, speed, sensitivity, and fluorophore compatibility. We examine the optical principles and design of these new instruments and their ability to see more detail with greater sensitivity--down to single molecules with tens of nanometers resolution. Nanoscopes have revealed transient intermediate states of organelles and molecules in living cells and have led to new discoveries but also biological controversies. We highlight common unifying principles behind nanoscopy such as the conversion of a subset of probes between states (ground or excited) and the use of scanning (ordered or stochastic). We emphasize major advances, biological applications, and promising new developments.
荧光成像方法推动或突破了分辨率的衍射极限（约 200nm），发展迅猛。这些超分辨率纳米显微镜技术包括：受激发射损耗（STED）、Pointillism 显微镜（[荧光]光激活定位显微镜/随机光学重建显微镜，或（F）PALM/STORM）、结构光照明显微镜、全内反射荧光显微镜（TIRFM）以及结合多种模式的技术。每种技术在横向和轴向分辨率、速度、灵敏度和荧光团兼容性方面都具有独特的优势。我们研究了这些新仪器的光学原理和设计及其以更高的灵敏度看到更多细节的能力——分辨率达到数十纳米，可检测到单个分子。纳米显微镜揭示了活细胞中细胞器和分子的瞬态中间状态，并带来了新的发现，但也引发了一些生物学争议。我们强调了纳米显微镜背后的一些常见统一原理，例如探针子集在（基态或激发态）之间的转换，以及扫描（有序或随机）的使用。我们强调了主要的进展、生物学应用和有前途的新发展。
