Box 1 Table 1.

Comparison of fluorescence microscopy techniques

Method	Measurables	Advantages	Limitations
FRAP [8, 9]	Average protein diffusion Mobile/immobile fraction	Standard on any confocal microscope	Measures average behavior of entire population Low spatial and temporal resolution Requires bleaching with high intensity laser light
FRET [1, 18, 19]	Distance between donor and acceptor labels	Typical range is 1–10 nm Reports interactions between two labeled proteins or conformational changes within a dual-labeled protein	Requires tagging of proteins with appropriate fluorophores Potential for false-negative results
Homo-FRET [21, 63]	Protein aggregation state - based on anisotropy measurements	Single class of fluorphore needed	Anisotropy measurements require special equipment
SPT [5, 8, 9, 24, 25]	Trajectories and diffusion of individual proteins Reveals different modes of motion (free, restricted, immobile)	nm spatial and ms time resolution Multi-color SPT allows for distinguishing between multiple protein species	Slower proteins are easier to track Generation of monovalent probes is non-trivial Low labeling density required
FCS/FCCS [28–30, 64, 65]	Protein diffusion coefficients Protein-protein interactions Protein concentration	Live cell studies	Requires special detectors Measurements are slow (>10s) and made at a fixed point in the cell Not applicable for immobile proteins Limited concentration range
ICS/ICCS [31]	Protein number density and aggregation state	Acquired with standard laser scanning confocal microscope or TIRF set-up Can be applied to samples of low or high concentration	Measures average protein behavior, subpopulations are not distinguished
RICS/ccRICS [31, 36, 66]	Provides a spatial map of protein mobility and protein interactions	Acquired with standard laser scanning confocal microscope Can measure fast dynamics (µs-ms) Live cell imaging	Scan rate must be comparable to the diffusion being measured Assumes diffusion of proteins in a homogeneous medium Immobile species may mask diffusing particles, proper filtering of the images can correct this
N&B/ccN&B [35, 67]	Brightness and number of molecules in each pixel	Live cell imaging Possible to determine immobile proteins Can be measured with standard laser scanning confocal microscope	Assumes that the intensity fluctuations are due only to fluorescent molecules Photobleaching needs to be accounted for
STED [52, 55,56, 68]	Super-resolution images (~ 40nm)	Live cell imaging possible	Requires expensive, specialized equipment
Localization Microscopy [48, 52, 53, 59–61]	Super-resolution images (down to 10 nm)	Live PALM has been achieved with 60 nm and 25 s resolution	Ultimate spatial resolution requires fixed samples Data acquisition time can be long (min to hrs)