Table 1. Mobility parameters obtained for different tracer molecules in vitro and in the nucleus of living cells.

	D (μm2 s−1)
msFCCS in vitro
TetraSpeck beads (0.1 μm diameter)	4.4±0.1*
QDot 525 streptavidin conjugate	31±1*
msFCCS in living cells (nucleus)
GFP1	32±3†
GFP3	14±2†
GFP5	11±1†
FRAP in living cells (nucleus)
RFP1	31±7‡,§
GFP3	15±4‡,§
GFP5	10±1‡,∥

FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; msFCCS, multi-scale fluorescence cross-correlation spectroscopy; RFP, red fluorescent protein.

Data were either obtained by msFCCS or FRAP with radial profile analysis. The results for TetraSpeck beads in vitro are in excellent agreement with the value D=(4.4±0.7) μm² s⁻¹ previously determined by dual-focus FCS and dynamic light scattering69. According to the measured diffusion coefficient of QDots, a hydrodynamic radius of r_H=(7.8±0.3) nm was calculated that matches the specification value of (8.8±1.3) nm given by the manufacturer (Invitrogen). msFCCS results in living cells agreed very well with FRAP experiments on the same length scale.

^*Diffusion constants obtained by auto-correlation analysis are reported, since results were independent on time and length scale.

^†Diffusion constants for msFCCS analysis with an effective distance of d_eff=1.2 μm.

^‡FRAP results for a bleach circle radius of w_b≈1.3 μm.

^§An immobile fraction of (1±1) % was found on the minute time scale.

^∥An immobile fraction of (6±1) % was found on the minute time scale.