High-dimensional super-resolution imaging of subcellular lipid membranes with structured illumination

Structured illumination microscopy (SIM) is widely used in biological research because of its doubled spatial resolution, fast imaging speed, and low photon-toxicity. Recently we demonstrated polarized SIM (pSIM) exploiting the polarization modulation in SIM systems and achieved super-resolution of fluorescent dipoles [1]. Here we further develop a six-dimensional super-resolution imaging technique based on structured illumination for the studying of subcellular lipidomics in vivo. We use one florigenic lipid dye, Nile Red, to universally stain all the lipid membranes and obtain super-resolution mapping of the morphology, polarity, and phase of different lipid membranes from fluorescence intensity, emission spectrum, and polarization modulation. We image and quantitatively mapped the lipid polarity and phase on up-to-eight cellular compartments, including the plasma membrane, nuclear membrane, ER, mitochondria, lipid droplet, lysosome, endosome, and Golgi apparatus.