Live-cell STAR image acquisition was performed with a Nikon
Ti-2 microscope equipped with a motorized stage, stage-top incubator to maintain 37 °C and 5% CO
2 (Tokai Hit, INUBG2SF-TIZB), ×60 1.49-NA objective, manual TIRF illuminator (Nikon, TI-LA-TIRF), 488 nm (Obis, 488-150 LS), and 647 nm (Obis, 1196627) excitation lasers, fiber coupling optics: fiber mount (Thorlabs, MBT621D), converging and directing the laser objective (Olympus,
RMS10X), optical fiber (Thorlabs,
P3-405BPM-FC-2), C-NSTORM QUAD 405/488/561/638 nm TIRF dichroic. Images were acquired with an
Optosplit III (Cairn Research) image splitter with ET525/50 m and ET705/72 m emission filters (Chroma), and T562lpxtr-UF2 and T640lpxtr-UF2 dichroic mirrors to split the fluorescence emission onto separate regions of the ORCA-Flash 4.0 v3 scientific complementary metal-oxide-semiconductor camera (Hamamatsu). The system was coupled by a data acquisition device (
NIDAQ, National Instruments, BNC-2115) and controlled using Nikon Elements software (version 5.02) and Coherent Connection software (version 3.0.0.8). Image acquisition was performed through NIS JOBS.
Optosplit III was calibrated using the manufacturer protocol and the NanoGrid (Miraloma Tech, LLC, A00020).
Nawara T.J., Williams YD I.I., Rao T.C., Hu Y., Sztul E., Salaita K, & Mattheyses A.L. (2022). Imaging vesicle formation dynamics supports the flexible model of clathrin-mediated endocytosis. Nature Communications, 13, 1732.