Fig. 4: Trifunction sphingomyelins (TFSMs) can be used for expansion microscopy (ExM).
a TFSMs are compatible with ExM. HeLa cells were incubated with TFSM 1 for 24 h in the presence of 1 % FBS. Samples were fixed, stained with BODIPY-FL-DBCO as well AlexaFluorTM (AF)546-azide, and then either imaged by conventional CLSM or 4-fold expanded. n = 3. Scale bars: 50 µm (~ 12.5 µm with 4-fold expansion factor). b, c Förster resonance energy transfer (FRET) can be measured in expanded samples. HeLa cells were incubated with TFSM 1 for 24 h in the presence of 1% FBS. Then, the compound was removed, and cells were treated with 2.5 µg/ml bacterial sphingomyelinase (bSMase) or left untreated for 24 h. Samples were fixed, stained, and expanded as described in (a) and analyzed via acceptor photobleaching to determine FRET efficiency. n = 4. d–f Lysosomes possess high SM content. HeLa cells were treated as described in (a), and lysosomal-associated membrane protein 1 (LAMP1) was stained by an anti-LAMP1 primary and a CF568 secondary antibody. Images recorded in the FRET channel were multiplied by factor 2 and divided by the BODIPY-FL (donor) channel resulting in images that describe the metabolic state of the molecule. n = 1. Scale bars: 50 µm (~ 12.5 µm with 4-fold expansion factor) and 12 µm for zoomed images (~ 3 µm with 4-fold expansion factor). Statistics: Two-sided unpaired Student’s t test (c). Bars represent means ± SD. n corresponds to biological replicates. Source data and detailed statistics are provided as a Source Data file.
