Fig. 4: SNAP-tag2 performance in live-cell super-resolution microscopy.
a, Comparison of HaloTag7, SNAP-tag2 and SNAPf-tag performance in CLSM and STED microscopy. HeLa cells stably coexpressing HaloTag7, SNAP-tag2 or SNAPf-tag in the mitochondria (Cox8a localization sequence) together with mEGFP (no specific localization) were labeled with CA–SiR or TF–SiR (100 nM) for 1 h and washed afterward. SNAP-tag2 and HaloTag7 show comparable performance in STED imaging, while SNAPf-tag shows insufficient signal under the same imaging conditions. This experiment was performed three times with similar results. Scale bars, 10 µm. Lookup tables: green (mEGFP) and red-hot (SiR). b,c, CLSM and STED images of U2OS cells stably expressing Vim–SNAP-tag2 (b) or Vim–SNAPf-tag (c) labeled with CF–SiR (100 nM) for 1 h. Cells were washed before imaging. White squares in the overview images (left) highlight the area chosen for magnification and STED imaging (right). The experiment was repeated twice with similar results. The FWHM of single intermediate filament fibers highlighted in b was determined to be 108 (±23) nm and 146 (±7.0) nm, underlining the higher resolution achieved with SNAP-tag2 compared to SNAPf-tag (15 individual filaments from three individual images per condition). Scale bars, 5 µm (overview) and 1 µm (magnification). c,d, CLSM and STED images of U2OS cells stably expressing SNAP-tag2 (c) in the lumen of the endoplasmic reticulum (CalR–KDEL) and (d) on the outer mitochondrial membrane (TOMM20) labeled with CF–SiR (100 nM) for 1 h, demonstrating the versatility of using SNAP-tag2 in different cellular compartments. Cells were washed before imaging. Scale bars, 5 µm (overview) and 1 µm (magnification). e, Dual-color CLSM and STED images of U2OS cells expressing Vim–SNAP-tag2 and LifeAct–HaloTag7. SNAP-tag2 was labeled with CF–SiR (100 nM) and HaloTag7 was labeled with CA–MaP618 (100 nM) for 1 h; cells were washed afterward. Scale bars, 5 µm (overview) and 1 µm (magnification). Lookup tables: cyan (SNAP-tag2–SiR) and orange-hot (HaloTag7–MaP618).
