Tie inverted microscope stand

To determine mitochondrial membrane potential, MEL 103 cells cultured on a glass bottomed 24 well culture plate at a density of 2X10⁴ were treated with the appropriate concentrations of the drugs overnight. Before the microscopic analysis the cells were incubated with 5 nM tetramethylrhodamine, methyl ester (TMRM) (Thermo Fisher). Time lapse images were obtained with a custom spinning-disk microscope built around a CSU-10 Confocal head (Yokogawa), a 897 Xion EMCCD camera (Andor), a 4-line monolithic laser launch (Agilent) and a TiE inverted microscope stand (Nikon) equipped with a stage top incubator that regulates CO2 at 5% and temperature at 37°C. The cells were imaged at an interval of 3 minutes for a total period of 3 hours.

A Nikon motorized TIRF illuminator mounted on a Nikon TiE inverted microscope stand was used. Laser excitation was fiber coupled from a four-line (405, 488, 561 and 638 nm) combiner (Oxxius).Emission was collected through dual pass filters from Chroma: blue/yellow-orange (420–480 nm / 570–620 nm) and green/far-red (505–550 nm / 650–850 nm). Imaging was performed through a 1.45 NA, plan apochromatic oil immersion objective (Nikon) using an Andor Zyla 5.5 sCMOS camera. For time-lapse imaging, up to 10 individual fields were marked using the motorized stage and imaged every 30 s. 0.5 ml of 5 µM Rapamycin (Fisher Scientific BP2963–1) + 150 nM GSK-A1 (Sigma SML2453) were added after 2 min to achieve bath concentrations of 1 µM and 30 nM, respectively.

A Nikon A1R-HD resonant scanning laser confocal microscope was used, mounted on a Nikon TiE inverted microscope stand. Images were collected through a 1.45 NA, plan apochromatic oil immersion objective (Nikon). Green (EGFP) and far red (iRFP713) fluorescence were co-excited using the 488 and 640 nm laser lines of a fiber-coupled LUN-V combiner, whereas red (mCherry) fluorescence was excited on a subsequent line scan to avoid cross talk, using the 561 nm laser line. Emission was collected using the following filters: green (505–550 nm), yellow-orange (570–620 nm) and far-red (650–850 nm). Confocal planes were collected with a pinhole of 1.2 Airy Units calculated for the far-red channel in resonant mode with 8 or 16 frame averaging. For time lapse imaging, up to 10 fields of cells were selected using the motorized stage and imaged every 30 s. Addition of rapamycin was accomplished by pipetting 0.5 ml of complete HEPES imaging media containing 5 µM rapamycin (Fisher Scientific BP2963–1) to the 2 ml already in the dish, achieving a final bath concentration of 1 µM.