Figure 3.

Genetic incorporation of unnatural amino acids (UAAs) as a tool to label proteins for NanoSIMS. A) Fluorescence and NanoSIMS images of a representative cell that had incorporated PRK in syntaxin 1, and was subsequently labeled with SK155. A confocal image of the Star635 fluorescence (left), a ¹⁹F NanoSIMS image, and an overlay (Star635 signal in red; ¹⁹F in green). Additional fluorescence and NanoSIMS images are shown: a ¹⁴N image, and a confocal image of immunostained calnexin, an endoplasmic reticulum marker. Scale bar: 2 μm. B) The ratio between ¹⁹F and ¹⁴N in non-transfected cells was measured and compared with cells expressing different proteins. It was significantly higher for all expressed proteins: SNAP-25 (p<0.01), syntaxin 1 (p<0.001), and syntaxin 13 (p<0.001; t-tests). The number of analyzed cellular regions is: 32 for non-transfected, 371 for SNAP-25, 281 for syntaxin 1, and 448 for syntaxin 13 expressing cells. C) Linear relationship between ¹⁹F/¹⁴N ratio in a cell and SK155 (Star635) intensity in the corresponding cell regions.