Bag Nirmalya, Wohland Thorsten
Departments of Biological Sciences and Chemistry, and NUS Center for Bio-Imaging Sciences (CBIS), National University of Singapore, 117557 Singapore; email:
Annu Rev Phys Chem. 2014;65:225-48. doi: 10.1146/annurev-physchem-040513-103641. Epub 2013 Dec 13.
Fluorescence fluctuation spectroscopy (FFS) techniques provide information at the single-molecule level with excellent time resolution. Usually applied at a single spot in a sample, they have been recently extended into imaging formats, referred to as imaging FFS. They provide spatial information at the optical diffraction limit and temporal information in the microsecond to millisecond range. This review provides an overview of the different modalities in which imaging FFS techniques have been implemented and discusses present imaging FFS capabilities and limitations. A combination of imaging FFS and nanoscopy would allow one to record information with the detailed spatial information of nanoscopy, which is ∼20 nm and limited only by fluorophore size and labeling density, and the time resolution of imaging FFS, limited by the fluorescence lifetime. This combination would provide new insights into biological events by providing spatiotemporal resolution at unprecedented levels.
荧光涨落光谱（FFS）技术能够在单分子水平上提供具有出色时间分辨率的信息。通常应用于样品中的单个点，最近它们已扩展为成像形式，即成像FFS。它们在光学衍射极限下提供空间信息，并在微秒至毫秒范围内提供时间信息。本综述概述了成像FFS技术已实现的不同模式，并讨论了当前成像FFS的能力和局限性。成像FFS与纳米显微镜的结合将使人们能够记录具有纳米显微镜详细空间信息（约20纳米，仅受荧光团大小和标记密度限制）以及成像FFS时间分辨率（受荧光寿命限制）的信息。这种结合将以前所未有的水平提供时空分辨率，从而为生物事件提供新的见解。
