All cell lines tested were stained immediately before imaging. Briefly, cells were trypsinized, counted and seeded in glass bottom μ-Dishes (Ibidi - Munich, Germany) at 2 × 104 cells/dish, and incubated for 24 h at 37°C in an atmosphere of 5% CO2. Cells were rinsed with Dulbecco's Modified Eagle Medium without phenol red (Euroclone) and loaded with 500 ηM NRB-AF12, 500 ηM ER-Tr red or green (#E34250 and #E34251; Thermo Fisher), and 500 ηM pHrodo red (#P10361; Thermo Fisher) at 10 μg/ml. In another experiment, LX2 cells were seeded in glass bottom μ-Dishes at 2 × 104 cells/dish and incubated for 24 h at 37°C in an atmosphere of 5% CO2. Cells were transfected with either 500 ηg of MitoDS-red or KDEL-red vector using Lipofectamine LTX Reagent (Invitrogen), according to the manufacturer specifications. At 24-h post-transfection, cells were stained with either 500 ηM NRB-AF12 or 500 ηM ER-Tr green. Cells were mounted in a Okolab microscope stage heated chamber warmed to 37°C. All images were collected with a Yokogawa CSU-X1 spinning disk confocal on a Nikon Ti-E inverted microscope equipped with a Plan Apo 60x NA 1.4 objective. Probes were excited with a 488 and 561 nm Lasers. To separate the individual emissions a dichroic filter 405/488/561/635-25 (Semrock, Rochester, NY) followed by a 440-40/521-21/607-34/700-45 filter (Semrock, Rochester, NY) were used and subsequently filtered further using band-pass filters (Semrock BP-525/30-25 and BP-607/36-25 for the green and red channels, respectively). Images were acquired with an Andor Technology iXon3 DU-897-BV EMCCD camera. For time-lapse experiments, images were collected every 10 s, using an exposure time of 500 ms.