Orca flash 4.0 camera

Yeast cultures were grown over night at 30 °C in YPD or SC media (buffered to pH 5.5 with 50 mM MES) to an OD_600nm of 0.2 to 1.2. Cells were harvested at 3000 × g, resuspended in 2–3 ml of fresh media at an OD_600nm of 0.1 to 0.2. FM4-64 was added from a 10 mM stock in DMSO to a final concentration of 10 μM and cells were incubated at 30 °C for 1 h. After washing two times with media, cells were shaken for 1.5 to 2.5 h at 30 °C in the same media as before, but without FM4-64.
For confocal imaging, we used a Perkin-Elmer UltraView Vox Confocal Spinning Disk Setup on an inverted Zeiss Microscope with a 100x oil immersion objective (NA 1.41) and two Hamamatsu ORCA-Flash 4.0 cameras. FM4-64 was visualized in fast acquisition mode with excitation by the 488 nm laser. For stack imaging, 9 focal planes with a distance of 0.5 μm were acquired at each time point. Non-confocal microscopy was performed on an inverted Leica DMI6000B fluorescence microscope equipped with a 100x/1.4 NA lens and a Hamamatsu C10600-10B camera (ORCA-R2). Images were processed using ImageJ. Stack projections were obtained using the maximum intensity function. Brightness and contrast were linearly adjusted. Exposure of the cells to excitation light was kept at a minimum in order to avoid bleaching and light-induced fusion of vacuoles, which can occur upon prolonged illumination.

PHIL pumps connected to PBS and 100 µM FITC (Sigma-Aldrich, F6377-100G) in PBS filled 96-well plate (Greiner-CELLSTAR^®-96-Well) wells with 100 µL PBS. PHIL then aspirated the well contents, added 100 µL 100 µM FITC, aspirated, and added 100 µL PBS over various periods and intervals. All volumes were pumped at 50 µL/s. FITC fluorescence was imaged on a Nikon Ti-Eclipse inverted microscope equipped with an Orca Flash 4.0 camera (Hamamatsu Photonics K.K.), using a ×10 objective (NA 0.45) (Nikon Instruments Europe B.V.).

Pipet positioning accuracy was conducted by filling a single pipet with 100 µM FITC (F6377-100G, Sigma-Aldrich) in PBS and positioning it in the center of well F12 in a 96-well plate (Greiner-CELLSTAR^®-96-Well) approximately 0.1 mm above the bottom of the well. FITC fluorescence within the pipet as it moved away from this location and returned was imaged every 1 s on a Nikon Ti-Eclipse inverted microscope that was equipped with an Orca Flash 4.0 camera (Hamamatsu Photonics K.K.), using a ×10 objective (NA 0.45) (Nikon Instruments Europe B.V.) and pipet location was recorded using custom scripts written for Youscope (www.youscope.org).

Pipet tips were primed by pumping liquids into them until a small volume was ejected. Pumped volume accuracies were determined by pumping 10, 25, 100, and 1000 µL of diH₂O onto the walls of empty 1.5 mL MaxyClear snaplock microcentrifuge tubes (MCT-150-A, Corning Inc.) which were weighed before and after to determine the true pumped volume (n = 10 repetitions per condition). Maximum flow rate calculations were determined by pumping 100 µL diH₂O onto the walls of empty PCR tubes at 10, 25, 50, and 100 µL/s which were weighed before and after to determine true volume pumped. Automated FITC dilution curves were generated by loading pipets with 100 µM FITC in PBS and PBS before pipetting 0–100 µM FITC solutions in 96-well plates (Greiner-CELLSTAR®-96-Well) in 10 µM increments. FITC fluorescence was imaged on a Nikon Ti-Eclipse inverted microscope equipped with an Orca Flash 4.0 camera (Hamamatsu Photonics K.K.), using a ×10 objective (NA 0.45) (Nikon Instruments Europe B.V.).