Methods of single-molecule fluorescence spectroscopy and microscopy

TLDR

Optical spectroscopy at the single‑molecule level has become a powerful technique for probing nanoscale behavior of molecules in complex environments, revealing hidden heterogeneity and dynamic state changes without ensemble averaging. This article reviews experimental techniques of single‑molecule fluorescence spectroscopy and microscopy, focusing on room‑temperature studies that track the same molecule over extended periods. Successful single‑molecule detection requires optimizing signal‑to‑noise, and the review details the physical parameters, four microscopic methods (epifluorescence, TIRF, confocal, near‑field scanning), and the diverse emission properties (polarization, spectrum, FRET, position) that can be recorded to extract dynamic information such as lifetimes, photochemistry, environmental fluctuations, enzymatic activity, and quantum optics. Single‑molecule spectroscopy and microscopy are emerging as valuable tools for studying dynamics in complex systems, especially where ensemble averaging or lack of synchronization obscures process details.

Abstract

Optical spectroscopy at the ultimate limit of a single molecule has grown over the past dozen years into a powerful technique for exploring the individual nanoscale behavior of molecules in complex local environments. Observing a single molecule removes the usual ensemble average, allowing the exploration of hidden heterogeneity in complex condensed phases as well as direct observation of dynamical state changes arising from photophysics and photochemistry, without synchronization. This article reviews the experimental techniques of single-molecule fluorescence spectroscopy and microscopy with emphasis on studies at room temperature where the same single molecule is studied for an extended period. Key to successful single-molecule detection is the need to optimize signal-to-noise ratio, and the physical parameters affecting both signal and noise are described in detail. Four successful microscopic methods including the wide-field techniques of epifluorescence and total internal reflection, as well as confocal and near-field optical scanning microscopies are described. In order to extract the maximum amount of information from an experiment, a wide array of properties of the emission can be recorded, such as polarization, spectrum, degree of energy transfer, and spatial position. Whatever variable is measured, the time dependence of the parameter can yield information about excited state lifetimes, photochemistry, local environmental fluctuations, enzymatic activity, quantum optics, and many other dynamical effects. Due to the breadth of applications now appearing, single-molecule spectroscopy and microscopy may be viewed as useful new tools for the study of dynamics in complex systems, especially where ensemble averaging or lack of synchronization may obscure the details of the process under study.