Polyunsaturated and saturated FFAs induce membrane phase separation. M109 cells were stained with membrane dyes DIOC18 (green) to probe liquid-ordered (Lo) phase and Rh-DOPE (red) to probe liquid-disordered (Ld) phase. (A) M109 cells exhibit uniform distribution of Lo and Ld phases. Yellow: overlaid of green and red signals. M109 cells pre-treated with (B) linoleic acids or (C) palmitic acids for 4 days exhibit membrane phase separation. (D) M109 cells pre-treated with oleic acids for 4 days exhibit uniform distribution of Lo and Ld phases. (E) CARS imaging can distinguish membrane phase separation induced by linoleic acids. Strong and weak CARS contrasts correspond to Lo phase, where cytoplasmic GFP and other cellular content reside, and Ld phase, where there is an absence of any cellular content, respectively. (F) CARS imaging cannot distinguish membrane phase separation induced by palmitic acids where cellular content, as marked by cytoplasmic GFP expression, is uniformly distributed. The final concentration of all FFAs used was kept at 50 μM to minimized lipotoxicity to M109 cells. Scale bars: 25 μm.