Ixon ultra 897 emccd camera

Cells were imaged at 37°C in an open chamber mounted on an inverted microscope N-STORM Nikon Eclipse Ti microscope with a 100x oil-immersion objective (N.A. 1.49). Fluo4-AM was illuminated using 472 (± 30) light from a diode. Emitted light was collected using an a 520 (± 35) nm emission emission filter. Time-lapse at 0.033 Hz were acquired with an exposure time of 100 ms for 990 s with an Andor iXon Ultra 897 EMCCD camera (image pixel size, 160 nm) using Nikon software. The analysis was performed on a section of the soma that was in focus at different time points. Fluorescence intensities collected in the soma before (F0) and following (F) bath addition of the drugs, were backgroundsubtracted. The data were analyzed using ImageJ (NIH, USA). Normalization of fluorescence intensity was performed by dividing the mean fluorescence intensity after drug application by the mean fluorescence intensity before drug application.

Unroofed cells were stained either with 16.5 pmol of Alexa Fluor 350-phalloidin (Life Technologies, A22281), Alexa Fluor 568-phalloidin (Life Technologies, A12380), or Alexa Fluor 647-phalloidin (Life Technologies, A22287) for 15 min depending on spectra of expressing FPs. Then cells were rinsed with PBS. 1 mm × 1 mm large montage was generated for proteins of interest and phalloidin using a Nikon Eclipse Ti inverted microscope with a 100×, 1.49 NA objective (Nikon, SR HP Apo TIRF) and an Andor iXon Ultra 897 EM-CCD camera under the control of Nikon Elements software. Images were obtained by TIRF illumination except for Alexa Fluor 350 which was imaged by epi illumination. This map was used to find the cells expressing the target proteins for FLIM and CLEM analysis. The imaged area was marked with a circle (4 mm in diameter) around the center of the imaged area using an objective diamond scriber (Leica, 11505059)^{40 (link),67 (link)}. The immersion oil was carefully removed from the bottom of the glass coverslip. The sample was subsequently imaged by FLIM or stored in 2% glutaraldehyde (Electron Microscopy Sciences, 16019) at 4 °C until EM sample preparation.