Fig. 4.

Differential nanoscale localization and dynamics of fatty acid analogs depend on metabolic state of cells. a Conventional fluorescence images of BODIPY-C₁₂ red exhibit co-localization with the ER protein Sec63-2 × GFP under fed condition. The SMLM image of BODIPY-C₁₂ red shows the ER membrane in high resolution and distinct spots on the ER below the optical diffraction limit. b The co-localization of BODIPY-C₁₂ red with BODIPY-NL shows that denser spots of BODIPY-C₁₂ co-localize with LDs. c The single-molecule tracking of BODIPY-C₁₂ red reveals predominantly free diffusion along the ER membrane. d Under fasted conditions, added BODIPY-C₁₂ red forms distinct puncta along the cell periphery that do not co-localize with Sec63-2 × GFP at the nuclear portion of the ER. The super-resolution images resolve puncta below the diffraction limit. e The distinct BODIPY-C₁₂ red puncta do not co-localize with LDs (BODIPY-NL) in fasting cells. f The single-molecule tracking of BODIPY-C₁₂ red exhibits mostly confined immobilization in fasting cells. g The MSD vs. time of BODIPY-C₁₂ red molecules shows free diffusion (D = 0.051 ± 0.013 μm² s⁻¹) under fed conditions (blue) and confined immobilization under fasted conditions (red) (Fed: 321 traces; Fasted: 151 traces; error: standard deviation from 6 movies each with > 3 cells). h The radial distribution function shows high-density clusters of BODIPY-C₁₂ red localizations upon fasting (red) and a much smaller fraction of clustered localizations in fed cells (blue) (Fed: 4219 localizations, 2 cells; Fasted: 4933 localizations, 2 cells). Scale bar: 1 µm unless specified