Table 2.

Fluorescence-based methods commonly used to analyze biomolecular interactions, stoichiometries and dynamics in the nanosecond to minute time range. Commonly, confocal fluorescence spectroscopy and total internal reflection (TIRF) spectroscopy are used to analyze the dynamics of single molecules and to measure intra- or intermolecular distances via FRET.

Fluorescence-Based Method		Time Scale	Advantages	Disadvantages
Confocal fluorescence spectroscopy	in solution	ns–ms	- molecule sorting for donor- and/or acceptor-labeled species via alternating laser excitation (ALEX) possible - measurements on freely diffusing and immobilized molecules possible	- limited concentration range of labeled species (5–200 pM) - time resolution is limited by the diffusion-time in the focus
	immobilized	ns–min	- fluorescence lifetimes measurements possible - kinetic information	- interactions with the surface due to immobilization might negatively influence activity of the biomolecule - sequential measurements
Total internal reflection fluorescence microscopy (TIRF)		ms–min	- molecule sorting for donor- and/or acceptor-labeled species via ALEX possible - parallel measurement of hundreds of individual molecules - spatial information on the observed molecules - able to monitor not synchronized or rare events	- interactions with the surface due to immobilization might negatively influence activity of the biomolecule - no fluorescence lifetime measurements possible