Dual response of HydroFlippers to changes in membrane tension^a.

	Probeb	dhic	dhhd	Δdhe (%)	τ 1i f (ns)	τ 1h g (ns)	Δτ1h (%)
1	1 (PM)	6.3	6.5	-3	4.8	4.4	8
2	1 (-C)i	6.1	—	8j	4.8	—	3k
3	2 (Lyso)	2.9	2.8	4	4.4	4.0	10
4	3 (Mito)	2.3	1.9	17	4.4	4.0	8
5	4 (ER)	1.8	1.5	17	4.3	3.7	15
6	4 (–C)i	1.1	—	39l	4.1	—	10m
7	5G (GA)n	2.5	2.3	8	4.2	3.8	10
8	5E (ER)o	1.7	1.2	29	3.8	3.7	5
9	1 (Lo)p	11	—	—	5.2	—	—
10	1 (Ld)q	1.2	—	—	3.4	—	—

^aFrom triexponential fit of FLIM images in HK cells (errors, see ESI).

^bFlipper (target MOI).

^cdh_i = area Στ_1i/area Στ_2i in FLIM histogram under iso-osmotic (i) conditions (e.g.Fig. 3f).

^ddh_h = area Στ_1h/area Στ_2h in FLIM histogram under hyper-osmotic (h) conditions.

^eFlipper hydration change in response to membrane tension: Δdh = (1 – dh_h/dh_i) × 100%.

^fFluorescence lifetime value of the slowest component from the fitted fluorescence decay under iso-osmotic (i) conditions (e.g.Fig. 2d).

^gSame as f, under hyper-osmotic (h) conditions.

^hFlipper planarization in response to membrane tension: Δτ₁ = (1 – τ_1h/τ_1i) × 100%.

ⁱMeasured after cholesterol (C) removal from cells with MβCD.

^jCompared to dh_i of 1 (6.6) in untreated cells measured on the same day.

^kCompared to τ_ih of 1 (5.0) in untreated cells measured on the same day.

^lAs j using 4 and compared to dh_i = 1.8.

^mAs k using 4 compared to τ_ih = 4.5.

ⁿMeasured in transiently transfected HK cells with ST-HaloTag-HA expressed inside GA.⁸⁰

^oMeasured in transiently transfected HK cells with HaloTag-Sec61B expressed inside ER.⁷⁸

^pMeasured in SM/C GUVs.

^qMeasured in DOPC GUVs.