All images were captured using an inverted fluorescence microscope (Eclipse Ti-E, Nikon) equipped with either an electron-multiplying charge-coupled device camera (ImagEM C9100-13, Hamamatsu; or iXon Ultra 897, Andor) or a scientific complementary metal-oxide-semiconductor camera (ORCA-flash 4.0 C11440-22C, Hamamatsu). We used a light-emitting diode light source (SPECTRA X Light Engine, Lumencor) to provide illumination with filter sets (from Semrock or Nikon): (i) excitation (Ex), 390/40 nm; dichroic, 405 nm; emission (Em), 452/45 nm (for DiFMU); (ii) Ex, 427/10 nm; dichroic, 458 nm; Em, 483/32 nm (for mseCFP); (iii) Ex, 480/40 nm; dichroic, 505 nm; Em, 535/50 nm (for mNeonGreen, and fluorescein); (iv) Ex, 504/12 nm; dichroic, 515 nm; Em, 542/28 nm (for mVenus); (v) Ex, 554/23 nm; dichroic, 573 nm; Em, 609/54 nm (for tdTomato, and mRuby2); and (vi) Ex, 630/38 nm; dichroic, 655 nm; Em, 694/44 nm. A 60× objective lens (Plan Apo VC; numerical aperture, 1.4; Nikon) was used for imaging experiments.

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