Spinning disc confocal microscope

Imaging of HeLa cells stably expressing mito-DsRed was performed using a spinning disc confocal microscope (Perkin-Elmer, Waltham, MA, USA) equipped with a 60x/1.4 NA oil-immersion objective using a 488 nm laser line for excitation. The cells were seeded on glass bottom 3 cm dishes (MatTek, Ashland, MA, USA) and maintained in full growth medium supplemented with 10 mM HEPES during imaging. Images were taken for a duration of 30 to 45 min in a chamber heated to 37 °C. Image analysis was performed using Fiji. 3D stacks were compressed with max projection and brightness was adjusted for optimal visibility of mitochondria. At least 20 cells per condition were analyzed in detail. At least 30 mitochondria per cell were manually measured for their major and minor axis. For branched mitochondria, only the longest path was measured. Mitochondria with a major axis of less than 0.5 µm were ignored since those signals could originate from mitochondria derived vesicles. Mitochondrial aspect ratio was determined by dividing minor axis by major axis for each mitochondrion and the arithmetic mean for each cell was calculated. Additionally, all imaged cells were categorized in to three categories (tubular, intermediate, and fragmented) after blinding by renaming files with random numbers.

To monitor the ΔΨ_m, melanoma cells or melanocytes were seeded on glass bottom dishes (Ø 3.5 cm, MatTek, Son, Netherlands) and incubated with 2.5 µM GP for 2 and 4 h, respectively, or with 10 µM carbonyl cyanide m-chlorophenyl hydrazine (CCCP), an oxidative phosphorylation uncoupler (Leblanc 1971 (link)), for 2 h as positive control. After incubation, cells were washed with PBS once and loaded with 100 nM of the mitochondrial membrane potential sensitive dye tetramethylrhodamine methyl ester (TMRM) and 100 nM of the mitochondrial membrane potential insensitive dye MitoTRACKER™ Green for 0.5 h at 37 °C. The accumulation of the fluorescent TMRM in intact mitochondria leads to a bright fluorescence signal which is weakened by adding apoptotic or other stressors, indicating a loss of mitochondrial membrane potential (Creed and McKenzie 2019 (link); Scaduto and Grotyohann 1999 (link)). Subsequently, cells were washed with PBS once and fresh medium was added to the cells. Cells were analyzed with a spinning disc confocal microscope (PerkinElmer, Waltham, Massachusetts, USA). At least 20 image stacks per sample were evaluated. The ΔΨ_m is calculated by TMRM/MitoTRACKER™ Green quotient and the mock-treated control was set at 100%.

Images were obtained using an upright fluorescent microscope (Axioskop2, Carl Zeiss, MicroImaging Incorporated, Thornwood, NY), a laser-scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) and/or a spinning-disc confocal microscope (PerkinElmer, Waltham, MA). Image analysis was conducted with the use of Zeiss LSM 510 and Volocity 2.0 and Adobe Photoshop software.

Cells were grown to 50% confluence on a coverslip, fixed in 4% paraformaldehyde for 20 min at 37 °C, and permeabilized with 0.15% Triton X-100 for 10 min. The cells were then blocked with 3% bovine serum for 30 min, incubated with primary antibodies overnight at 4 °C, and then stained with secondary antibodies for 1 h at room temperature. Samples were observed under a Zeiss LSM-710NLO confocal fluorescent microscope equipped with a 100 × /1.42 NA oil immersion objective lens, a Zeiss LSM 880 Laser Scanning Microscope equipped with a 63 × /1.40 NA oil immersion objective lens using the Airyscan function, or a GE Healthcare DeltaVision OMX SR imaging system (GE) equipped with a 63 × /1.40 NA oil immersion objective lens using 3D-SIM function.
For time lapse observation, cells were transfected with mCherry-KDEL, treated with 50 μM eto 8 h after transfection, and observed under a spinning disc confocal microscope (PerkinElmer) equipped with a 100 × /1.40 NA oil immersion objective lens. Images were captured at 5-min intervals. Noise was removed from the captured images using Volocity software (PerkinElmer).

HAP1 cells expressing matrix-targeted GFP were used to study the mitochondrial morphology on a PerkinElmer spinning disc confocal microscope equipped with a 60× oil objective (NA = 1.49) and a chamber maintaining 37°C and 5% CO₂. Images were acquired with a Hamamatsu C9100 camera having dimensions of 1,000 × 1,000 pixels after excitation at 488 nm. The cells were classified as tubular, intermediate, and fragmented depending on the majority of mitochondria present in a cell belonging to a particular class. Cells classified as intermediate class contained a mixture of predominantly short pieces, few tubular or fragmented mitochondria, whereas cells classified as tubular and fragmented contained mostly long tubular and very short fragments of mitochondria, respectively.