Table 1.

Methods for studying single molecule nucleosome structures and dynamics

Method		Advantages	Limits
Fluorescence	Fluorescence methods in general	Low level of system perturbation High temporal resolution Native environment	No control over system dynamics Limited fluorophore labeling strategies Artifacts due to fluorophores
	Single molecule FRET (smFRET) in general	No need for calibration for internal comparison (ratiometric measurement) High spatiotemporal resolution	One-dimensional measurement
	smFRET with surface-immobilized nucleosomes	Efficient to study detailed dynamics of a complex process with more than 2 states Easy to deconvolve heterogeneous populations (i.e. easy to filter out contaminants and inactive species) Limit of temporal resolution:  with wide-field imaging: a few milliseconds  with one point detection: tens to hundreds of microseconds (limited by photon emission rate)	Possible effects of surface interactions with nucleosomes
	smFRET and/or FCS with diffusing nucleosomes	No effects of surface interactions with nucleosomes FCS: Efficient to test if fast dynamics exist (tens to hundreds of microseconds)	Impractical or impossible to study detailed dynamics of a complex process with more than 2 states Impractical or impossible to monitor dynamics with time scales longer than diffusion of nucleosomes
Force	Force methods in general	Direct control of system dynamics Direct measure of interactions Native environment	Low level of control over reaction coordinate High level of system perturbation Limited force-handle labeling strategies
	Optical tweezers	High force resolution (sub-pN)	Inefficient data collection (typically one molecule at a time)
	Magnetic tweezers	Efficient data collection in a wide-field imaging setup (multiple molecules at a time)	Higher level of noise (i.e. limited precision in controlling and/or measuring force at a rate higher than a few Hz)
Scanning probe		Multi-dimensional imaging	Mostly under non-native conditions Typically very low or no temporal resolution