HeLa cells were cultured on #1.5 coverslips. After 24 hours cells were transiently transfected using PolyEthylene Imine (PEI) using 1 μg of DNA and 3 μg of PEI per well on a 6-well plate, with a plasmid bearing LifeAct tagged with the yellow fluorescent protein variant Venus. After 24 hours cells were washed briefly with PBS and fixed with PFA for 10 min at room temperature. Samples were extensively washed with PBS and imaged in the presence of PBS.
Imaging of the samples was carried out on a Leica SR-GSD microscope. Images were taken in TIRF mode at 100 frames per second. The setup consisted of the following components: an inverted microscope (DMI6000 B, Leica Microsystems GmbH), a 1.47-NA TIRF objective (HCX PL APO 100× NA 1.47), a tube lens providing an extra factor of 1.6× in magnification, a488-nm fiber laser (2RU-VFL-P-300-488), a 532-nm fiber laser (2RU-VFL-P-1000-532-B1R, MPB Communications), a 642-nm fiber laser (2RU-VFL-P-1000-642-B1R, MPB Communications) and an EMCCD camera (iXon DU-897, Andor) with an effective EM gain of 148. Images were taken in TIRF mode at 100 frames per second for ~5100 time frames, giving a total measurement time of about 1 min for each color. Colors were imaged in decreasing wavelength order. The filter cube (642HP-T) for imaging with the 642-nm laser consisted of an excitation filter (zet405/642x), a dichroic mirror (t405/642rpc) and emission filters (et710 100lp and ET650LP). The epifluorescence filter cube (532HP-T) for imaging with the 532-nm laser consisted of an excitation filter (zet405/532x), a dichroic mirror (t405/532rpc) and emission filters (et600/100 m and ET550LP). Pixel size in the image was 93.11 nm.
Imaging of the samples was carried out on a Leica SR-GSD microscope. Images were taken in TIRF mode at 100 frames per second. The setup consisted of the following components: an inverted microscope (DMI6000 B, Leica Microsystems GmbH), a 1.47-NA TIRF objective (HCX PL APO 100× NA 1.47), a tube lens providing an extra factor of 1.6× in magnification, a488-nm fiber laser (2RU-VFL-P-300-488), a 532-nm fiber laser (2RU-VFL-P-1000-532-B1R, MPB Communications), a 642-nm fiber laser (2RU-VFL-P-1000-642-B1R, MPB Communications) and an EMCCD camera (iXon DU-897, Andor) with an effective EM gain of 148. Images were taken in TIRF mode at 100 frames per second for ~5100 time frames, giving a total measurement time of about 1 min for each color. Colors were imaged in decreasing wavelength order. The filter cube (642HP-T) for imaging with the 642-nm laser consisted of an excitation filter (zet405/642x), a dichroic mirror (t405/642rpc) and emission filters (et710 100lp and ET650LP). The epifluorescence filter cube (532HP-T) for imaging with the 532-nm laser consisted of an excitation filter (zet405/532x), a dichroic mirror (t405/532rpc) and emission filters (et600/100 m and ET550LP). Pixel size in the image was 93.11 nm.
