Fig 1.
Comparison of live cell labelling with picoGreen and SYBR Gold at various dilutions.
A549 cells were incubated with Hoechst 33342 and picoGreen or SYBR Gold at indicated dilutions and imaged. Representative fields of view of the labeled cells in the “green” channel. LSM880 Airyscan Fast, 20x/Air objective, scale bar 50 μm. Single optical sections. White squares mark the areas shown at a higher zoom to the right of the entire 423×423 μm fields of view. For 1:1000 and 1:2000 dilutions, the same images are shown at two brightness settings: the “default” brightness settings optimized for 1:10000 dilution, and the “reduced brightness” settings adjusted to minimize saturation.
Fig 2.
Distribution of the cytoplasmic to nuclear signal ratio in the living cells labelled with picoGreen or SYBR Gold.
Nine 423×423 μm fields of view per condition were used for the quantification. The ratio of the integrated cytoplasmic and nuclear signals was calculated for each cell (n = 841…2769). Normalized distributions of these ratios are shown. Median value for each distribution is shown in the upper right corner. Note different scale on y axis for 1:10000 dilution.
Fig 3.
Effect of incubation duration and concentration on SYBR Gold localization in live cells.
Indicated dilutions of SYBR Gold in DMEM were added to live HeLa cells in simultaneously; cells were kept with SYBR Gold for 1 hour or for 2 days (as indicated on the Figure). Then, cells were stained with Hoechst 33342 and imaged in parallel (i.e. all samples were imaged 2 days after the start of incubation). LSM880 confocal microscope; 20x/Air objective; scale bar 100 μm. 415×415 μm fields of view are shown. Further details are described in Materials and Methods.
Fig 4.
Co-localization of SYBR Gold with TFAM.
Live HeLa cells transiently expressing TFAM-mEos2 and stained for 30 min. with SYBR Gold (final dilution 1:10000) and Mitotracker Deep Red™ (final concentration 250 nM). Images (z-stacks) of live cells were acquired and used for analysisPS1 Elyra, confocal mode, 100x/1.46 Oil objective; single optical slice from a representative field of view is shown; scale bar 10 μm. Red dashed squares on the left panels mark the region of interest which is shown at higher magnification in the right panels.
Fig 5.
Effect of FCCP on SYBR Gold localization.
HeLa cells were pre-incubated with FCCP and then labelled with SYBR Gold (dilution 1:10000) and Mitotracker Deep Red in the presence of FCCP. Merged SYBR Gold (green) and Mitotracker Deep Red (magenta) channels are shown. White squares mark regions of interest which is shown on zoomed images. Individual confocal slices. LSM880; 63x/1.4 Oil objective.
Fig 6.
Effect of fixation and permeabilization on SYBR Gold localization in the cells.
HeLa cells were stained with SYBR Gold (stock diluted 1:10000) and Mitotracker CMXRos Red (250 nM) for 30 min. Single optical slices acquired on spinning disk microscope; sequential acquisition; scale bar 10 μm. Other conditions are further described: A. Live HeLa cells stained with SYBR Gold; B. HeLa cells stained alive with SYBR Gold and then fixed with 2% PFA in PBS for 30 min; C. HeLa cells stained alive with SYBR Gold, then fixed with 2% PFA in PBS for 30 min and permeabilized with 0.1% Triton X100 for 15 min; on the lower C panel, the same image is shown, but brightness in green channel is set higher; D. HeLa cells fixed with PFA, then permeabilized with 0.1% Triton X100 for 15 min. and finally stained with SYBR Gold and Mitotracker CMXRos Red. To acquire images shown on panel D, EMCCD gain of the camera for green channel was reduced by factor of 12 in comparison to A-C, to avoid overexposure.
Table 1.
Effect of SYBR Gold labelling under various conditions on cell viability.
Fig 7.
Comparison of confocal (cyan) and SIM (yellow) imaging of live cells labelled with SYBR Gold.
A. Representative images (single optical slices). The same objective 100x 1.46 Oil TIRF and the same sampling (50 nm/pixel) is used for both LSM and SIM images. Left column shows entire field of view; Scale bar 10 μm; ROI is marked with red dashed line; ROI is shown in the right column with higher magnification (scale bar 1.25 μm). B. An intensity profile across two nucleoids (marked as a red arrow on higher magnification panels) in confocal (orange) and SIM (cyan) channels. C. Intensity profiles of fluorescent bead (red) and mitochondrial nucleoids (cyan) in SIM images. Further, we acquired SIM 3D datasets of live cells co-stained with SYBR Gold and Mitotracker Deep Red. Mitochondrial nucleoids appeared as bright spots within mitochondria, frequently as rows with equal distances between neighbors (Fig 8A). Distribution of distances between neighbor nucleoids showed a single maximum at 450 nm approx. (Fig 8B). This agrees with the values of 400–800 nm previously reported for fixed mammalian cells (Fig 1E in [45]). Interestingly, peaks in SYBR Gold channel intensity profiles correspond to local minima on the intensity profiles in Mitotracker Deep Red channel (Fig 8A, bottom panels), suggesting that freely diffusing mitochondrial matrix proteins are excluded from nucleoids; this agrees with previously reported exclusion of a mitochondria marker YFP-tagged cytochrome oxidase from nucleoids [45]. Thus, SIM imaging of SYBR Gold-stained cells enabled us to reproduce in live cells the observations of mitochondrial nucleoids, previously done in fixed cells and with diffraction-limited microscopy.
Fig 8.
Quantification of mitochondrial nucleoids positions in live HeLa cells on SIM images.
A. Representative optical slice of a z-stack of live cells labelled with SYBR Gold (green) and Mitotracker Deep Red (magenta). PS1 Elyra, SIM mode. Red dashed square on the top panels mark a region of interest which is shown on the bottom panels. Scale bar, 10 μm. Intensity profile for a linear ROI is shown on bottom zoomed panels. B. Intensity profile across image of a cell (single optical slice) described above. SYBR Gold signal, green line; Mitotracker Deep Red signal, magenta line. C. Histogram of distribution of distances to the closest neighbors for mitochondrial nucleoids in 3D SIM datasets spanning whole cell thickness.
Fig 9.
Nucleoid tracking on live SIM images.
Representative images of a field of view of DMSO-treated cells. Top: two-color SIM image taken before acquisition of the time lapse series. Green, SYBR Gold channel; magenta, mitotracker channel. Bottom: Nucleoids tracks from 50-frames SIM time series, in SYBR Gold™ channel (S1 and S2 Videos are examples); frame time 1.8 s; tracking by Imaris 8.4.1 software. Tracks are color-coded by maximal track speed (μm/s); PS1 Elyra, SIM mode, 100x/1.46 Oil objective; scale bar 10 μm.
Fig 10.
Effect of microtubule depolymerization on the nucleoids motions.
A. Control of microtubules depolymerization upon nocodazole treatment. Maximum intensity projections of SIM z-stacks of live HeLa cells stained for mitochondrial DNA (SYBR Gold), mitochondria (Mitotracker Deep Red) and microtubules (transiently expressed MAP4-mCherry construct), without (top) and with (bottom) nocodazole treatment. Scale bar, 10 μm. B-D. Mitochondrial nucleoids tracks parameters in the absence (blue bars) and in the presence (orange bars) of nocodazole. Histograms show distribution of track mean speed (B), track maximal speed (C) and track length (D). Particle tracking is described in Materials and Methods section.
Table 2.
Effect of microtubule depolymerization on nucleoids motions parameters.