. 2019 Oct 10;9:14527. doi: 10.1038/s41598-019-50742-8

Table 1.

Translocation reference levels for cytoplasm- and membrane-targeted fluorescent control constructs measured through the EGFP, mCherry, and FRET-FLIM imaging detectors (supporting methods) at different pixel sizes.

Pixel size (nm)	Cytoplasm targeted			Membrane targeted
	Free-diffusing mCherry-6Gly-EGFP			Lyn₁₁-FKBP₂-mRFP
	EGFP PM/Cyt intensity	mCherry PM/Cyt intensity	FRET-FLIM $f_{D}^{e f f}$ PM/Cyt	mCherry channel PM/Cyt intensity
275	0.84 ± 0.01	0.85 ± 0.01	1.04 ± 0.01	2.2 ± 0.1
68	0.99 ± 0.01	0.98 ± 0.01	—	2.7 ± 0.1

275 nm pixel-size images were acquired for FRET experiments (Figs 2–5), while 68 nm pixel-size images were acquired for the quantification of PtdIns(3,4,5)P₃ levels (Fig. S6). The difference between PM/Cyt intensity values for 275 nm and 68 nm pixel size is due to the lower resolution of the former. $f_{D}^{e f f}$ PM/Cyt is the PM to cytoplasm $f_{D}^{e f f}$ ratio, which quantifies the relative distribution of FRETting pairs. The uncertainty is indicated as SEM for at least N > 20.