Cell culture and CoQ₁₀ incubation were performed as previously described [19]. Briefly, primary human dermal fibroblasts (HDF) obtained from ten different adult donors (n = 10) were purchased from Alphenyx (Marseille, France), TebuBio (Offenbach, Germany), and Tissue Solutions Ltd (Glasgow, United Kingdom). Isolated HDF were tested negative for mycoplasma and all cultured in a humified incubator at 37 °C and 5% CO₂. Cells were seeded in 35 mm microscopy culture dishes (Greiner Bio-One GmbH, Frickenhausen, Germany) with 60,000 to 100,000 cells per dish. The cell culture medium consisted of DMEM 4.5 g/L D-glucose, 10% fetal bovine serum (FBS), 1% penicillin G (100 units/mL), 100 µg/mL streptomycin (P/S), and 1% 100X GlutaMAX™ (all cell culture reagents from Gibco™, Thermo Fisher Scientific, Waltham, MA, USA). CoQ₁₀ (Kaneka Corporation, Takasago, Japan) was dissolved using a mixture of glycerol and the emulsifier PEG-40 hydrogenated castor oil (CoQ₁₀:glycerol:PEG-40 = 0.4:0.6:1) according to Knott et al. [5]. The CoQ₁₀ stock solution was added to DMEM medium 24 h after seeding and once the cells were ~70% confluent. The final CoQ₁₀ concentration was 15 µg/mL. The cells were further incubated for 24 h, and then FLIM images were acquired. Untreated cells and cells treated with the respective solvent control were used as controls. Prior to FLIM measurement, the medium was replaced with Tyrode’s buffer consisting of 135 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 5 mM glucose (all chemicals from Sigma-Aldrich, St. Louis, MO, USA). The pH was kept at 7.4.

Following the experimental protocol by Kalinina et al. [20], FLIM imaging was performed using a Zeiss LSM 710 NLO (non-linear-optic) laser scanning microscope (Zeiss, Oberkochen, Germany) equipped with a femtosecond pulsed Mai Tai AX HPDS titanium-sapphire laser (Spectra Physics, Santa Clara, CA, USA) with a repetition rate of 80 MHz and a tuning range of 690 nm to 1040 nm. The excitation wavelength for NADH FLIM was 740 nm. The laser pulse for two-photon excitation was controlled by an electronic system, enabling time-correlated-single-photon-counting (TCSPC). With this, signal detection was accomplished through an HPM-100-40 hybrid detector system (Becker & Hickl GmbH, Berlin, Germany) coupled to the non-descanned detection (NDD) port of the microscope. FLIM images were acquired using the SPC64 software (Becker & Hickl GmbH) with a resolution of 512 $\times$ 512 pixels. Images were analyzed using the SPCImage software (v8.1, Becker & Hickl GmbH). The fluorescence decay curve of NADH was fitted to an incomplete two-component exponential model:

(1)$$I(t)=a_1{e}^{-t/\tau l}+{a_2}^{e-t/\tau 2}$$

I(t) is the fluorescence intensity, a1 and a2 are the relative amplitudes, $\tau$₁ and $\tau$₂ are the fluorescence lifetimes of the two components. Fixed values of $\tau$₁ = 400 ps and $\tau$₂ = 2500 ps corresponding to the free and protein-bound fractions of NADH and an incomplete exponential model were used for the fluorescence decay fitting. Based on the fitting applied to each pixel, a colormap was generated where the fluorescence lifetime corresponds to a color legend.

The phasor plot was calculated with the SPCImage software (v8.1, Becker & Hickl GmbH). For the metabolic pattern segmentation, discrete lifetime ranges of 600–800 ps, 800–1000 ps, and 1000–1200 ps were selected.

Segmented images were generated with OriginPro, and pixel areas were quantified using FIJI (https://fiji.sc) [21]. The number of pixels in the discrete lifetime ranges was related to the total number of pixels within the whole lifetime range (area A). The mean area (A_mean) was calculated as the mean of area A from six different images from the same donor. This procedure was completed using two individual donors.

Fibroblasts for EM imaging were prepared along with the FLIM samples. Carbon-coated glow-discharged sapphire disks were placed in cell culture dishes [22]. The seeding and treatment of the cells was conducted as described above. After the addition of CoQ₁₀, samples were high-pressure frozen using a Wohlwend HPF Compact 01 high-pressure freezer (HPF) (Engineering Office M. Wohlwend GmbH, Sennwald, Switzerland) following the protocol of Villinger et al. [22]. Samples were freeze substituted following the protocol of Walther and Ziegler [23] with a substitution medium consisting of acetone (100% analytically grade) with 0.2% osmium tetroxide (ChemPur, Karlsruhe, Germany), 0.1% uranyl acetate (Merck KGaA, Darmstadt, Germany) and 5% water, and subsequently embedded in epoxy resin. Sectioning was performed using an Ultracut (UCT) ultramicrotome (Leica Microsystems, Wetzlar, Germany) equipped with a 35° diamond knife and a trim diamond (https://www.diatomeknives.com). For TEM imaging, 70 nm thick sections were mounted on copper slot grids. For STEM tomography, sections of 700 to 900 nm were mounted on a glow-discharged copper grid with parallel bars. A droplet of 10% (w/v) poly-L-lysine (Sigma Aldrich) in water was added. The sections were then dried at 37 °C for 5 min. After that, 15 µL of a colloidal gold solution (particle size: 15 nm, https://aurion.nl) diluted 1:2 in water was applied to both sides of the sections. The gold nanoparticles were later used as fiducial markers for tomogram reconstruction. A 5 nm carbon coating was applied on both sides with a BAF 300 electron beam evaporation device (https://www.opticsbalzers.com). Samples were imaged using a JEOL JEM 1400 transmission electron microscope (JEOL, Tokyo, Japan) for an overview of the cellular ultrastructure and quantification purposes. Tomogram acquisition was performed with a JEOL JEM-2100F scanning transmission electron microscope (JEOL, Tokyo, Japan) at 200 kV acceleration voltage using the EM-Tools-software (TVIPS, Tietz, Germany). A series of 97 bright-field images with 200,000$\times$ magnification was acquired at tilt angles from –72° to +72° with a 1.5° increment. The illumination time per image was 22 s. Each image had 1024 $\times$ 1024 pixels, with a pixel size of 3.4 nm. For reconstruction and segmentation of the tomograms, the IMOD package [24], v4.9.12 was used. Tomograms were reconstructed as described previously [25].

The statistical analysis of the data was performed using the software OriginPro (OriginLab Corporation, Northampton, MA, USA). For each condition, ten adult fibroblast donors (n = 10) were used to estimate the mean and median fluorescence lifetimes. For each donor, 12 NADH fluorescence lifetime images were analyzed. The Shapiro–Wilk test was used to evaluate whether the data sets were normally distributed. The one-way analysis of variance (ANOVA) test was used to test the significance of the population means between a pair of test conditions. In addition, a nonparametric Wilcoxon signed rank test determined the significance of the population medians. All hypothesis tests were performed using a significance level of 5% (p = 0.05).

It is well known that CoQ₁₀ is a crucial component of the mitochondrial respiratory chain. In order to characterize the metabolic state under the influence of CoQ₁₀, the mean fluorescence lifetime ($\tau$_mean) was investigated in human dermal fibroblasts (HDF) supplemented with CoQ₁₀. Changes in the fluorescence lifetime of NADH were detected using TCSPC FLIM. For each of the n = 10 donors, the lifetime was calculated for 12 images. Fig. 1 shows FLIM images of the solvent control (a,b) and the CoQ₁₀-treated cells (c,d) of a representative donor. An average overall n = 10 donors yielded a mean NADH fluorescence lifetime of $\tau$_mean = 841 $\pm$ 17 ps for the solvent control cells. In comparison, the HDF treated with 15 µg/mL CoQ₁₀ showed a significantly longer mean NADH fluorescence lifetime of $\tau$_mean = 858 $\pm$ 16 ps (p = 0.03) (Fig. 1e). A longer mean NADH fluorescence lifetime of the CoQ₁₀-treated cells indicated a metabolic shift of dermal fibroblasts toward more oxidative phosphorylation and thus more aerobic energy production. We did not observe a component with a lifetime greater than 3000 ps, which would correspond to bound NADPH.

Fig. 1.

NADH FLIM images of primary fibroblasts in false colors (representing lifetimes from 600 ps in orange to 1100 ps in blue) from one representative donor. (a,b) solvent control cells. (c,d) cells treated with 15 µg/mL coenzyme Q₁₀ (CoQ₁₀) for 24 h. Blue signals corresponding to longer nicotinamide adenine dinucleotide (NADH) lifetimes are found more in (c) and (d) than in (a) and (b), which indicates a stimulation of cellular energy metabolism. The scale bars represent 20 µm. (e) Mean and median NADH fluorescence lifetimes of CoQ₁₀-treated human dermal fibroblasts (HDF) vs. solvent control and untreated control based on the NADH fluorescence lifetime imaging microscopy (FLIM) images of fibroblasts of n = 10 different donors. The mean and median NADH fluorescence lifetimes of CoQ₁₀ and solvent control are significantly different, with a p-value of 0.03. *, significant differences are marked with an asterisk.

Phasor-based image segmentation of cells from two donors was performed to identify metabolically active intracellular areas. For each of the two donors, six images were segmented with three discrete lifetime ranges (600–800 ps, 800–1000 ps, 1000–1200 ps), and the area A covered by the signals in the respective lifetime range was quantified for each individual image (Fig. 2), and the mean area A_mean was calculated for all images. Signals from the lifetime range of 600–800 ps were found in large areas of the cells, covering the nuclei and cytoplasm (Fig. 2). A_mean occupied by signals from the short lifetime range of 600–800 ps was smaller in CoQ₁₀-treated cells than solvent control cells (A_mean = 30% for the 1000–1200 ps range vs. A_mean = 21% for the 600–800 ps range in one donor and A_mean = 35% for the 1000–1200 ps range vs. A_mean = 29% for the 600–800 ps range for the second donor), sometimes leaving empty spaces in the cytoplasm around a clearly distinguishable nucleus (Fig. 2). In one of the donors, signals from the longer lifetime range of 1000–1200 ps clearly occupied a larger area in CoQ₁₀-treated cells (A_mean = 9% vs. A_mean = 17%), whereas the second donor only showed a trend in this direction. The phasor-based segmentation highlights more intracellular areas with long NADH lifetimes (1000–1200 ps) after CoQ₁₀ treatment, indicating stimulated mitochondria.

Fig. 2.

Example of the calculation of metabolically active areas (A) for one image of one donor. Phasor-based metabolic pattern segmentation of solvent control cells and 15 µg/mL CoQ₁₀- treated cells from one representative donor after 24 h. False colors represent lifetimes from 600–800 ps in red and 1000–1200 ps in blue. A indicates the area covered by the signal in the respective lifetime range. The signal from the short lifetime range is absent from the cytoplasm of some CoQ₁₀-treated cells, and more signal from the long lifetime range is present in the cytoplasm, possibly representing activated mitochondria. The scale bars represent 20 µm.

The mitochondrial ultrastructure was investigated with transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography. TEM analyses of fibroblasts from two donors were performed, and for each condition, 12 cells were analyzed. The TEM images of solvent control cells showed dark, dense granules within some mitochondria (Fig. 3a,b). In general, mitochondria were darkly stained and found near the sites of the endoplasmic reticulum (dark arrowheads in Fig. 3a). A descriptive quantification showed that the granules were highly abundant in the solvent control cells. In one donor, the dark granules were found only in two untreated control cells (16.6% of a total of 12 cells). In the cells treated with the solvent control, the granules appeared in three cells (25%). Upon CoQ₁₀ stimulation, the granules were found only in one cell (8.3%), and the majority of mitochondria exhibited well-defined and parallel cristae (Fig. 3c). For this donor, the number of mitochondrial granules decreased along with an increase in the NADH lifetime upon CoQ₁₀ stimulation. The increase is visible in the rendered FLIM image (Fig. 1c,d), where the fibroblasts appeared predominantly blue, corresponding to longer NADH lifetimes. The findings obtained for the donor shown in Fig. 3 were confirmed by the TEM images and quantification for a second donor, where the granules were found in 0 untreated cells, five cells treated with the solvent control, and one cell treated with CoQ₁₀.

Fig. 3.

TEM images of primary fibroblasts from one representative donor. (a) Transmission electron microscopy (TEM) image showing several mitochondria from a solvent control cell. Numerous black granules appear in some mitochondria, while others (marked with a white star in (a) and (b)) appear with no granules but stain brighter. Endoplasmic reticulum (ER)-contact sites of mitochondria with black granules (black arrows) are often encountered. (b) Higher magnification image of two mitochondria marked with the white rectangle in (a). (c) Mitochondrion from a CoQ₁₀-treated cell (15 µg/mL CoQ₁₀ for 24 h). Cristae are regularly arranged, and no granules are visible. The scale bars represent 500 nm in (a) and 200 nm in (b,c).

From virtual sections obtained from a STEM tomogram (Fig. 4) of a solvent control cell, the dark granules were well visible, similar to the TEM images. Manual segmentation of the tomogram showed that the granules were present over the entire volume of the mitochondria (Fig. 4a,b; Video S1). As visible from the segmentation and virtual sections, the contact sites of the endoplasmic reticulum (ER) (segmented in green) with mitochondria were present and spanned over large areas near the mitochondrial surface. In CoQ₁₀-treated cells, the mitochondria appeared vastly connected, as shown in the rendered segmentation of a STEM tomogram (Fig. 4c; mitochondria segmented in blue). Mitochondria exhibited well-pronounced cristae on virtual sections (Fig. 4d) and well-visible contact sites with the endoplasmic reticulum (green in Fig. 4c).

Fig. 4.

STEM tomogram of a solvent control cell (a,b) and a CoQ₁₀- treated cell (15 µg/mL CoQ₁₀ for 24 h) (c,d). (a) Segmented scanning transmission electron microscopy (STEM) tomogram showing the stress granules also found in TEM images (compare Fig. 3). Stress granules are segmented in violet, mitochondria in blue and endoplasmic reticulum in green. (b) 3D segmentation of only stress granules (in violet), showing a homogenous distribution in the mitochondria. (c) Segmentation of the mitochondria (light blue) without stress granules and endoplasmic reticulum (green) in a CoQ₁₀-treated cell. (d) Virtual section of a tomogram of a CoQ₁₀- treated cell showing healthy mitochondria (compare Fig. 3c). The scalebar represents 500 nm in (a–d).

In summary, complementary examination of mitochondrial ultrastructure in control cells revealed multiple stress granules, whereas mitochondria from CoQ₁₀-treated cells showed predominantly well-defined and parallel cristae with minimal stress granules.