By superlocalizing the positions of millions of single molecules over many camera frames, a class of super-resolution fluorescence microscopy methods known as single-molecule localization microscopy (SMLM) has revolutionized how we understand subcellular structures over the past decade. In this review, we highlight emerging studies that transcend the outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at single-molecule and super-resolution levels. By encoding/decoding high-dimensional information—such as emission and excitation spectra, motion, polarization, fluorescence lifetime, and beyond—for every molecule, and mass accumulating these measurements for millions of molecules, such multidimensional and multifunctional super-resolution approaches open new windows into intracellular architectures and dynamics, as well as their underlying biophysical rules, far beyond the diffraction limit.Expected final online publication date for the Annual Review of Physical Chemistry, Volume 75 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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活细胞中物理化学参数的单分子光谱和超分辨率绘图

通过在许多相机帧上对数百万个单分子的位置进行超定位，一类被称为单分子定位显微镜（SMLM）的超分辨率荧光显微镜方法彻底改变了我们在过去十年中理解亚细胞结构的方式。在这篇综述中，我们重点介绍了超越 SMLM 提供的出色结构（形状）信息的新兴研究，以单分子和超分辨率水平提取和绘制活哺乳动物细胞中的理化参数。通过对每个分子的高维信息（例如发射光谱和激发光谱、运动、偏振、荧光寿命等）进行编码/解码，并对数百万个分子的这些测量值进行质量累积，这种多维和多功能超分辨率方法开辟了新的方法。细胞内结构和动力学及其基本生物物理规则的窗口，远远超出了衍射极限。《物理化学年度评论》第 75 卷的预计最终在线出版日期为 2024 年 4 月。请参阅 http://www.annualreviews。 org/page/journal/pubdates 了解修订后的估计。