**Conflict of interest**

washing, the fresh culture medium was added and cells were treated for 4 h with 0.01% DMSO (vehicle control) or 7 μM Vac. Imaging was performed by IncuCyteZOOM® and red/green total fluorescence per image was quantified and collected using IncuCyteZOOM® software. The ratio between red and green fluorescence was calculated. A shift from red to green fluorescence

Sapphire discs (Engineering Office M. Wohlwend GmbH, Switzerland) were used as support for the adherent glioma cell culture. The 0.170 mm thick sapphire discs were carefully immersed in complete medium and the cells were seeded on top. A 50 μm gold spacer ring (diameter 3.05 mm, central bore 2 mm; Plano GmbH, Germany) was mounted in between two sapphire discs with the cells grown on them, similar to the protocol introduced by Hawes et al. [19]. These sandwiches were high-pressure frozen without aluminum planchettes and without the use of hexadecene. Freeze substitution was performed as described in Villinger et al. [20] with a substitution medium consisting of acetone with 0.2% osmium tetroxide, 0.1% uranyl acetate, and 5% water for 19 h. During this time period, the temperature was exponentially raised from 183 to 273 K. After substitution, the samples were maintained at room temperature for 30 min and then washed twice with acetone. After stepwise embedding of the samples in Epon (Fluka.com, USA) (polymerization at 333 K within 72 h), they were cut using a microtome (Leica Ultracut UCT ultramicrotome) with a diamond knife

and therefore, a decline in the red/green ratio indicates mitochondrial depolarization.

(Diatome, Switzerland) to semi-thin sections with a nominal thickness of 550 nm.

back-projection algorithm to form the tomogram [20].

**3.8. Statistical analysis**

**Acknowledgements**

For STEM tomography, 550 nm semithin sections were coated with 25 nm gold particles serving as fiducial markers for alignment of the images to a tomogram. The tilt series was collected on a JEM-2100F electron microscope operated at an acceleration voltage of 200 kV in the scanning transmission mode. Electron micrographs were recorded with a bright-field detector at a pixel size of 2.74 nm. A tilt-series was recorded from −72° to +72° at increments of 1.5°. The tomogram was created out of 97 images using the IMOD software package. Images were first aligned to an image stack and then computationally reconstructed using a weighted

Statistical analysis was performed using GraphPadPrism v7 (Graphpad.com, USA). To identify significant differences of endpoint analyzes, an unpaired two-tailed t-test was used. P-values are given in the respective graphs (column with the treatment). In case of kinetics, two-way ANOVA followed by Sidak's correction for multiple comparison. Multiplicity adjusted p-val-

We thank the International Graduate School in Molecular Medicine, Ulm University for supporting and evaluating HM, PS, and PSch. We are grateful to IntechOpen publisher for generous support to publish a virtual section of the supplementary file 1 in reference [16], as

ues are given. Observations with p < 0.05 were considered as significant.

**3.7. Electron microscopy**

90 Glioma - Contemporary Diagnostic and Therapeutic Approaches

The authors declare no conflicts of interest.
