Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

The protocol permits the specific labeling of mitochondrial nucleoids in live cells and the quantitative study of their motion at a high resolution. Incubation with the fluorescent dye only bypasses the need for fluorescent protein over expression, avoiding related artifacts and limitations and allowing our protocol to be applied to non-transfectable cells. To achieve preferential nucleoid staining, one should use SYBR Gold and nothing else under the conditions that we have optimized.

Otherwise, total cellular DNA may be stained. One day before the labeling procedure, culture four times 10 to the fifth HeLa cells in two milliliters of medium in a 35-millimeter Petri dish. The next morning, wash the cells in two milliliters of PBS before adding one milliliter of phenol red free culture medium and one milliliter of the appropriate 2X labeling solution.

After 30 minutes at 37 degrees Celsius and 5%carbon dioxide, carefully aspirate the dye containing supernatant from each 35-millimeter Petri dish and wash the cells with two milliliters of PBS. Then feed the cells with fresh phenol red free cell culture medium and return the culture to the cell culture incubator protected from light until live imaging. For live cell imaging, at least an hour before the imaging session, place the stage top incubator onto the super resolution structured illumination microscope stage and set the temperature to 37 degrees Celsius and the carbon dioxide concentration to 5%Switch on all of the components of the microscope including the lasers and select a high magnification, high numeric aperture immersion objective as recommended for Super resolution structured illumination microscopy by the microscope manufacturer.

When the lasers have warmed up, install the 35-millimeter Petri dish onto the microscope stage and use the oculars to locate an area of interest with cells attached to the bottom of the dish. To acquire super resolution structured illumination microscopy images, use a back end high end electron multiplying charge coupled device camera. In the image acquisition software, set a high electron multiplying gain as recommended for the camera.

Before acquiring the time lapse series for nucleoid tracking, acquire a two color super resolution structured illumination microscopy image of the same field of view in one channel for mitochondrial staining and another one for SYBR gold. Set the mitochondrial image color channel to the appropriate excitation and emission for the mitochondrial stain used and set the appropriate excitation and bang Pass emission filter for the SYBR gold dye. Set the lowest laser power possible for both channels.

If the microscope acquires channels only sequentially, switch off the channel for the mitochondrial stain detection. untick the Z-stack box in the software to switch off the Z-stack acquisition to set the acquisition of a single focal plane and set the shortest possible camera exposure time. Set three rotations of the grid and acquire two dimensional images of the labeled cells at several laser power values and several exposure times to optimize the laser power in camera exposure time.

Select the laser power and camera exposure times that yield structured illumination microscopy images with bright spots in the mitochondria with little or no artifacts and start the acquisition of the time lapse series using the optimized settings. For analysis of the time lapse images, open the converted structured illumination microscopy images and an appropriate image analysis software that has a spot tracking module and click add new spots to start up the spots creation wizard. Under the create tab, click track spots over time and proceed to the second step of the wizard.

Set the estimated x-y diameter to 0.1 to 0.15 micrometers. Click background subtraction, and proceed to the third step in the wizard. Drag the vertical line in the histogram to adjust the threshold of the quality filter until the majority of the nucleotides are detected as spots in the artifacts are not detected within each frame.

Proceed to the fourth and fifth steps of the wizard and select the autoregressive motion algorithm. Set the max distance to 0.5 micrometers and the max gap size to zero. When the fine tuned parameters allow the software to detect all of the spots and build tracks correctly, Click the arrow now button to confirm creation of the tracks and click the statistics icon to extract the tracks statistics.

Then select the necessary statistics parameters and click Save As to export the values as dot CSV files for quantification and visualization. Labeling with high concentrations of SYBR gold or PicoGreen results in abundant staining of the nuclei and punctate staining within the cytoplasm. At lower concentrations, faint SYBR gold signal appears within the nuclei in a pattern of bright spots is observed within the cytoplasm.

PicoGreen labeling at low concentrations yields mostly nuclear staining. Simultaneous staining with low concentrations of SYBR gold in a far red mitochondrial dye reveals that nearly all of the SYBR gold staining occurs within the mitochondria while labeling at high concentrations results in significant staining of the nuclei and cytoplasm. Time-Lapse imaging reveals that after 45 minutes the nucleoid staining is close to saturation.

Fixation causes a slight redistribution of the dye to the nucleus, while permeablization of the fixed cells eliminates the dotted staining pattern of the mitochondria and enhances the nuclear staining. If SYBR gold is added to the cells after fixation and permeablization, then the dye's distributed uniformly across the cytoplasm and the nucleus resulting in the loss of the staining specificity. Live cell 3D imaging by super resolution structured illumination microscope reveals the mitochondrial nucleoid as bright spots within the mitochondria.

Tracking the positions of the nucleoid at a resolution beyond the diffraction limit demonstrates that the majority of nucleoids exhibit short distance random motions that are probably confined to the mitochondrial network. It is important to note that this protocol is not compatible with the fixation of the sample. However, our protocol should be compatible with a wide range of live cell imaging based assays for study of morphology, dynamics and activities of molecules and organelles.

Our study illustrates the advantages of retargeting non-organic dyes for cell based techniques. It may inspire the exploration of other fluorescent dyes for their application in cellular studies.