Table 2.

Summary of the most relevant contributions on the use of genetically encoded FPs as inert tracers of the intracellular environment. The spatial and temporal scale of reference is reported for each selected study, as well as the peculiar FP used, the biologic system chosen and a brief summary of the methodological approach employed, and results obtained.

Spatial Scale (nm)	Temporal Scale (ms)	Inert Tracer	Biologic System	Method/Result	Ref.
>1 µm	milliseconds	GFP	CHO cell cytoplasm	FRAP: GFP diffusivity is estimated by fitting the recovery curve. Result: GFP diffusion is 3–5-fold more suppressed than dilute solutions. This is interpreted as the result of macromolecular crowding	Refs. [21,22]
>1 µm	milliseconds	Dendra2	COS7 cell cytoplasm	PIPE: GFP diffusivity is estimated by fitting the time expansion of the photoactivated spot. Result: GFP diffusion is 3–5-fold more suppressed than dilute solutions	Ref. [27]
>1 µm	µ-to-milliseconds	GFP	CHO cell cytoplasm	RICS: GFP diffusion is extracted from raster-scan images averaging over the entire image. Result: GFP diffusion is 3–5 fold more suppressed than dilute solutions	Ref. [44,45]
>1 µm	milliseconds	GFP	CHO cell cytoplasm	SPIM-iMSD: GFP diffusion is measured on a grid of points simultaneously. iMSD analysis yields the average GFP diffusion law within the intracellular environment. Result: GFP diffusion is 3–5-fold more suppressed than dilute solutions	Ref. [56]
200–300 nm	µseconds	GFP	Cell cytoplasm	Single point FCS: local measurement of GFP concentration and diffusion. Result: GFP diffusion is still 3–5-fold more suppressed than dilute solutions	Ref. [33]
200–300 nm	µseconds	GFP, GFP multimers	Cell nucleus	Single point FCS in multiple locations: GFP diffusion is measured on a grid of points, consecutively. Result: GFP diffusion is suppressed; no correlation is found between GFP diffusivity and chromatin density	Ref. [57]
From 200–300 nm to several microns	Hundreds of µseconds	GFP	CHO cell nucleoplasm	pCF analysis on a line: GFP diffusion is measured on a grid of points along a scanned line. Result: cross-correlation of points highlights disconnected GFP flow across chromatin density barriers	Ref. [58]
200 nm	50 µs	GFP, GFP3, GFP5, RFP	U2OS cell cytoplasm and nucleus	Multiscale fluorescence cross-correlation spectroscopy: GFP diffusion is measured on a grid of points simultaneously. Cross-correlation of points is used to measure the GFP transit time to reach the different locations. Result: the regulation imparted by the intracellular structural organization on FPs diffusion is characterized	Ref. [42]
80–100 nm	µseconds	GFP	CHO cell cytoplasm	Single point STED-FCS: GFP diffusion is measured, locally, at a sub-diffraction scale. Result: GFP diffusion is on the average only 2-fold more suppressed than dilute solutions, but spatial heterogeneity is highlighted	Ref. [41]
From 100 nm to whole cell	milliseconds	GFP	MB231 cell cytoplasm	2D-pCF analysis on SPIM-based measurements: GFP diffusion is measured on a grid of points simultaneously. The 2D-pCF algorithm enables to draw GFP molecular paths across space. Result: a map of the intracellular connectivity/obstacles to diffusion can be drawn	Ref. [59]
From 20–30 nm to several micrometers	1 µs	GFP, GFP2	CHO cell cytoplasm and nucleus	RICS-iMSD at tunable timescales: GFP displacement is measured by averaging over many microns at tunable time scales. Result: GFP unobstructed (Brownian) motion is observed below 100 nm, anomalous and then suppressed diffusion (3–5-fold) above 100 nm	Ref. [48]
<5 nm	5–50 ns	GFP	CHO cell cytoplasm	Anisotropy decay: GFP rotational diffusion is measured at the nanoscale. Result: GFP rotation is almost unhindered in the cell cytoplasm.	Refs. [21,22]