Tcs sp5 dm6000 cs

Live cell imaging was performed with an upright confocal laser scanning microscope (Leica TCS SP5 DM-6000 CS, Leica Microsystems, Vienna, Austria) and the connected LAS AF Software v4 (Leica Microsystems, Vienna, Austria). All images were taken with a 63× water immersion objective (NA 1.2). We used a multi-argon laser and selected a wavelength of 488 nm for excitation of the different fluorochromes. The emission wavelengths of GFP-ER (495–550 nm), FM4-64 (575–640 nm) and chlorophyll (670–770 nm) were detected by three different multipliers simultaneously. The focal depth was set to about 0.5 µm and the pinhole was adjusted to one airy disc. To obtain high resolution images without motion blurring, a scanning speed of 200 or 400 Hz were chosen. Single images were edited with FIJI software [31 (link)]. Optical sectioning in the z-direction was performed in the same resolution as in the x–y direction and resulted in z-stacks of 100–300 images, which were consecutively used to generate 3D reconstructions of the detected fluorescent cell structures.

All the observations were performed on living cells with an upright confocal laser scanning microscope (Leica TCS SP5 DM-6000 CS, Leica Microsystems, Vienna, Austria) and LAS AF Software v4 (Leica Microsystems, Vienna, Austria). The images were obtained by a 63× water immersion objective (NA 1.2). For the excitation of the different fluorophores, we used a multi-argon laser and selected the wavelength of 488 nm for excitation. Three different photomultipliers simultaneously detected the emission wavelengths of GFP (495–640 nm), FM4-64 (575–640 nm), and chlorophyll (670–770 nm), respectively. The pinhole was set to one airy disc and the focal depth was about 0.5 µm. The scanning speed was set between 200 and 400 Hz. For observations of acute plasmolysis, it was increased to 700 Hz. Z-stacks of 20–300 images were used as maximum projection or videos, and single images were processed and further investigated with FIJI software [22 (link)].

For confocal microscopy experiments, BT474 cells were plated on 35-mm Petri dishes (15,000 cells/cm²) and treated with appropriate drugs as indicated in the figure legends and/or in the text. Cells rinsed three times with 2 mL of HBSS buffer were incubated in 2 mL of HBSS buffer, supplemented with 2 µg/mL Hoechst 33342 (H3570, Invitrogen, Waltham, MA, USA), SYBR Green I at dilution of 1:200,000 (S7563, Invitrogen, USA), and 150 nM MitoTracker Deep Red 633 (M-22426, Molecular Probes, Eugenius, OR, USA) at 37 °C for 30 min in a CO₂-free thermostat. Following staining, the cells were rinsed 3 times with dye-free HBSS, and fluorescent images of cells were obtained using fluorescent scanning confocal microscope Leica TCS SP-5 DM6000 CS (Leica Microsystems, Wetzlar, Germany) at a sequential scanning mode using HCX PL APO lambda blue 63×, NA = 1.4 (Leica Microsystems, Wetzlar, Germany). Excitation and emission were set for Hoechst 33342 405 nm/460 nm, SYBR Green I 488 nm/540 nm, and MitoTracker Deep Red 633 633 nm/710 nm. Quantification of the number of mitochondrial nucleoids and mitochondrial size was performed using the fluorescence confocal images of BT474 cells labeled for mtDNA (stained with SYBR Green-1) and mitochondrial mass (stained with MitoTracker Deep Red). Images were analyzed using the Fiji software algorithm [52 (link)] and macros described in [12 (link)].