Ixon x3897 camera

NeuroBurst Orange Lentivirus (Sartorius, Germany) was used as a genetically-encoded calcium indicator. Virus transduction was performed on day in vitro (DIV) 10 with 3 µl of NeuroBurst. On DIV17, glass coverslips were prepared in an imaging chamber with Tyrode buffer (400 µl) and imaged at 37 °C with an inverted Nikon Ti eclipse epifluorescence microscope (Nikon, Japan). This set-up has an HBO-100 W lamp, a 20X Plan Apo (Nikon, Japan) objective, an IXON X3897 camera (Andor, UK), and an Okolab cage-incubator (Italy). Stimulation was performed with the addition of 400 µl high K⁺/low Na⁺ Tyrode buffer (70 mM KCl, 59 mM NaCl, 2 mM CaCl₂, 1 mM Mg₂Cl, 30 mM D-Glucose and 25 mM HEPES) on top of the initial 400 µl of Tyrode buffer. This solution was not removed till the end of the recording.

Cells and tissues were visualized using a Leica microscope (model DMR, Melville NY) fitted with 40X (PL Fluotar, NA 1.0) and 63X (PL APO, NA 1.32) objectives. Images on the DMR microscope were acquired using an Orca 100 CCD camera (model C4742-95; Hamamatsu, Bridgewater, NJ) and analyzed using ImageJ software (NIH version 2.0.0) or Metamorph. For N-SIM analysis, the samples were illuminated with spatially high-frequency patterned excitation light (100X objective lens, NA 1.49; TiE N-SIM microscope [Nikon] and iXON X3 897 camera [Andor Technology]). Images were reconstructed and analytically processed to reconstruct subresolution structure of the samples using Elements version 4 software (Nikon). For siNEDD8 and siCOPS3 EGFR internalization, the Axioimager and the Apotome2 was used with the 40x objective and a MIP of the Z sections was used.

We imaged fixed samples using an Olympus IX71 microscope (Olympus, Hamburg, Germany), equipped with a TIRF objective (100x, 1.45 NA) from Olympus. We used a F-View II CCD camera (1376 × 1032 pixels; pixel width 6.45 μm). Alternatively, we used a normal 60x objective (1.35 NA, Olympus). Imaging filters were: for green fluorescence 480/40 HQ excitation, 527/30 HQ emission, 505 LP Q beam splitter (catalogue number F41-054; from AHF, Tübingen, Germany). For red fluorescence: 620/60 HQ excitation, 700/75 HQ emission, 660 LP Q beam splitter (catalogue number F41-054; from AHF, Tübingen, Germany).
For live imaging we used a Nikon Ti-E epifluorescence microscope (Nikon Corporation, Chiyoda, Tokyo, Japan), equipped with a Plan Apochromat objective (100x 1.4 NA oil-immersion, Nikon). Excitation was performed using a HBO-100W mercury lamp. We used a IXON X3897 camera (512 × 5 12 pixels; pixel width 16 μm; Andor, Belfast, Northern Ireland, UK). The microscope was climate-controlled using an OKOLab system (OKOLab, Ottaviano, Italy). Imaging filters were: for green fluorescence, excitation filter 470/40 nm, emission filter 525/50 nm and dichroic 495 nm. For red fluorescence: excitation filter 545/25 nm, emission filter 605/70 nm and dichroic 565 nm.