Figure 6.

Dual FLIM/PLIM Imaging of KPC Cells Stably Expressing the Eevee-Akt-mT2 Intramolecular FRET Biosensor, Treated with Oxygen-Sensitive Nanoparticles

(A) A schematic illustration of the methodology applied to modify the multiphoton FLIM detection system to detect oxygen-sensitive PLIM through modulation of the EOM.

(B) A schematic demonstrating the use of a glass coverslip to induce a hypoxic response in vitro.

(C) qRT-PCR analysis of relative mRNA expression of hypoxia response genes upregulated in cells incubated for 2 hr in hypoxia (5%, 1%, and 0.1% oxygen) or under a coverslip, compared to normoxia, normalized to Rplp0 (n = 5). Mean ± SEM.

(D) Dual FLIM/PLIM imaging of Akt activity in KPC cells treated with MitoImage NanO2 and incubated with a glass coverslip for 1 or 2 hr (n = 4). Scale bars, 50 μm; insets, 10 μm. Mean ± SEM. p values are from a Student two-tailed parametric t test in all panels.

^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. See also Figures S5 and S6, and Tables S1 and S2.