Uplansapo 100x 1.40 oil objective

Epifluorescence microscopy to visualize GFP and stained samples was routinely carried out using an IX81 motorized inverted microscope (Olympus) equipped with a UPlanSApo 100X/1.40 Oil objective (Olympus). Excitation of fluorescently-labelled proteins and lipid droplets was carried out using a VS-LMS4 Laser-Merge-System with solid-state lasers (488 nm/50 mW). The laser intensity was controlled by a VS-AOTF100 System and coupled into the light path using a VS-20 Laser-Lens-System (Visitron System). Images were captured using a Charged-Coupled Device camera (Photometric CoolSNAP HQ2, Roper Scientific). All parts of the system were under the control of the software package MetaMorph (Molecular Devices).

STED nanoscopy was performed using a quad scanning STED microscope (Abberior Instruments, Göttingen, Germany) equipped with a UPlanSApo 100x/1,40 Oil objective (Olympus, Tokyo, Japan). The pinhole was set to 0.7–1.0 Airy units. A pixel size of 20–25 nm was used.
For live STED imaging, SNAP-Cell SiR was excited at 640 nm and STED was performed at 775 nm wavelength. PicoGreen was excited at 485 nm wavelength. The fluorescence signal was detected using avalanche photo diodes with bandpass filters. For STED imaging of SNAP-Cell SiR, a gating of 0.75–8 ns was applied. Dwell times of 7–10 µs were used. For STED images, each line was scanned 4 to 8 times and the signal was accumulated. For the confocal images, each line was scanned once.
For dual color STED imaging of fixed cells, STAR RED was excited at 640 nm and STED was performed at 775 nm wavelength. AlexaFluor 594 was excited at 561 nm wavelength. The fluorescence signal was detected using avalanche photo diodes with bandpass filters and a gating of 0.75–8 ns was applied. Dwell times of 10 µs were used. Each line was scanned 3 times and the signal was accumulated.

For immunofluorescence experiments, cells plated on coverslips 24 h before the experiment were fixed using 4% formaldehyde in PBS for 20 min at 37°C. Coverslips were washed three times with PBS and cells were permeabilized in 0.1% Triton in PBS for 15 min at room temperature. After three washes with PBS, coverslips were blocked in PBS containing 5% BSA for 30 min, incubated with primary antibodies for 1 h at room temperature, washed three times with PBS, and incubated with Alexa-conjugated secondary antibodies (1:2,000) and DAPI (1:2,000) for 30 min at room temperature. Coverslips were washed three times with PBS and mounted with Fluromount-G (Thermo Fisher Scientific). Cells were imaged with Olympus IX83 microscope connected with Yokogawa CSU-X confocal scanning unit, using UPLANSAPO 100x/1.40 Oil objective (Olympus) and Andor Neo sCMOS camera. Images were processed in Fiji (Schindelin et al, 2012 (link)).