Name: specific labeling of mitochondrial nucleoids for time-lapse structured illumination microscopy
Uploaded: 2020-06-04T13:00:00
Description: Watch this Scientific Journal Video about Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy at JoVE.com

The authors acknowledge Asifa Akhtar and Angelika Rambold (both Max Planck Institute for Immunobiology and Epigenetics) for providing HeLa cells.

NOTE: All the cell lines mentioned here were cultured in high glucose Dulbecco&#39;s Modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), glutamine, penicillin/streptomycin, and pyruvate. Equilibrate all media and supplements to be used on the day of labeling and imaging by warming them up to 37 &#176;C in an incubator set to 5% CO2. All cell culture work including labeling takes place in sterile conditions under a laminar flow hood.
1. L...

<ol>
	<li>Kaufman, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+transcription+factor+TFAM+coordinates+the+assembly+of+multiple+DNA+molecules+into+nucleoid-like+structures.">The mitochondrial transcription factor TFAM coordinates the assembly of multiple DNA molecules into nucleoid-like structures.</a> Molecular Biology of the Cell. 18 (9), 3225-3236 (2007).</li><li>Farge, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+sliding+and+DNA+denaturation+are+essential+for+DNA+organization+by+human+mitochondrial+transcription+factor+A.">Protein sliding and DNA denaturation are essential for DNA organization by human mitochondrial transcription factor A.</a> Nature Communications. 3, 1013 (2012).</li><li>Bogenhagen, D. 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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AAA(+)+protein+ATAD3+has+displacement+loop+binding+properties+and+is+involved+in+mitochondrial+nucleoid+organization.">The AAA(+) protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization.</a> Journal of Cell Biology. 176 (2), 141-146 (2007).</li><li>Ashley, N., Harris, D., Poulton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+mitochondrial+DNA+depletion+in+living+human+cells+using+PicoGreen+staining.">Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining.</a> Experimental Cell Research. 303 (2), 432-446 (2005).</li><li>Jevtic, V., Kindle, P., Avilov, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SYBR+Gold+dye+enables+preferential+labeling+of+mitochondrial+nucleoids+and+their+time-lapse+imaging+by+structured+illumination+microscopy.">SYBR Gold dye enables preferential labeling of mitochondrial nucleoids and their time-lapse imaging by structured illumination microscopy.</a> PLoS One. 13 (9), e0203956 (2018).</li></ol>

There are several critical components to the protocol: To achieve preferential labeling of mitochondrial DNA, the concentration of the DNA binding dye during incubation should be kept very low (e.g., a 1:10,000 dilution of a typical commercial stock), and the incubation time should be 30 min. The incubation time should never exceed 1 h. SYBR Gold dye should be used; other DNA-binding dyes are not bright enough to generate a strong signal upon labeling at a low concentration.
The limitation of ...

Circular 16.5 kbp DNA molecules constitute the genetic material of mitochondria, encoding 22 tRNAs, 2 rRNAs, and 13 polypeptides needed for mitochondrial oxidative phosphorylation complexes. Mitochondrial DNA bound to mitochondrial transcription factor a (TFAM) and several other proteins form the mitochondrial nucleoids1,2,3,4. Mitochondrial nucleoids moveand redistribute between the components of the mitochondrial network5,6 during its morphological remodeling, fission or fusion depending on cell cycle phase, stress, and other factors (reviewed in Pernas et al.7). In addition, the motion of mitochondrial nucleoids, is implicated in systemic lupus erythematosus disease8 and may play a role in other diseases. Fluorescence microscopy is a straightforward technique for live-cell studies of organelles, but the technique has a resolution of &#62;200 nm, which is larger than the size of mitochondrial nucleoids (~100 nm9,10,11,12). This limit has been circumvented by so called &#34;super-resolution&#34; techniques, such as stimulated emission depletion (STED) and single molecule localization microscopy (SMLM)13,14. So far, mitochondrial nucleoids and other DNAs were imaged in live cells by direct stochastic optical reconstruction microscopy (dSTORM)15. Fine sub-mitochondrial structures with positions correlating with mtDNA were observed by STED in live cells16. However, these super-resolution techniques require high illumination intensity, which causes phototoxic effects on living cells17. Therefore, time lapse imaging of mitochondrial nucleoids with resolution beyond diffraction limit is challenging. To address this, we used super-resolution structured illumination microscopy (SR-SIM)18. SIM requires a much lower illumination power dose than STED and SMLM19. Furthermore, in contrast to STED and SMLM techniques, SIM permits straightforward multicolor three-dimensional (3D) imaging, and it does not require particular photophysical properties of the fluorophores or imaging buffer composition19.
The conventional strategy for labeling mitochondrial nucleoids in live cells is fluorescent tagging of a mitochondrial nucleoid protein, such as TFAM20. However, in many cases, this strategy is not suitable. Moreover, overexpression of fluorescent protein-tagged TFAM produces a serious artifact21. Labeling of DNA with organic dyes has advantages over a fluorescent protein (FP)-based strategy. Organic dyes are free of constrains related to FP tagging: they can be used for any type of cells or tissueand can beapplied at any time point of an experiment. Live cell imaging of mitochondrial nucleoids has been reported with several DNA-binding dyes: DAPI22, SYBR Green23, Vybrant DyeCycle24, and picoGreen15,25,26. A substantial drawback of most DNA-binding dyes for nucleoid labeling is that they stain all DNA within the cell. Targeting a dye solely to mitochondrial DNA is highly desirable. To achieve that, careful selection of a dye possessing suitable physico-chemical properties is necessary. Lipophilic dyes possessing delocalized positive charge, such as rhodamine 123, are known to accumulate in live mitochondria, which preserve their negative membrane potential. In addition, an ideal dye for specific labeling of mitochondrial nucleoids should bind DNA with high affinity and emit bright fluorescence upon DNA binding. Considering these requirements, certain cyanines are promising (e.g., picoGreen), but nuclear DNA is abundantly stained by these dyes simultaneously with mitochondrial DNA15,25,26. The present protocol describes specific labeling of mitochondrial nucleoids in live cells with another cyanine dye, SYBR Gold (SG), and tracking of the nucleoids in time lapse super-resolution SIM videos. Moreover, SG-stained live cells can be imaged by any type of inverted fluorescent microscope (confocal, spinning disk, epifluorescence, etc.) suitable for living cells and equipped with a 488 nm light source.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Elyra PS1</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>multi-modal super-resolution microscope containing module for super-resolution structured illumination microscopy (SR-SIM)</td>
		</tr>
		<tr>
			<td>high glucose DMEM</td>
			<td>GIBCO/ThermoFisher</td>
			<td>31966021</td>
			<td></td>
		</tr>
		<tr>
			<td>ibidi 35-mm dish, glass bottom</td>
			<td>Ibidi Gmbh</td>
			<td>81158</td>
			<td></td>
		</tr>
		<tr>
			<td>ibidi 8-well microSlide, glass bottom</td>
			<td>Ibidi Gmbh</td>
			<td>80827</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris 8.4.1</td>
			<td>Bitplane/Oxford Instruments</td>
			<td></td>
			<td>image porcessing and visualisation software package</td>
		</tr>
		<tr>
			<td>iXon 885</td>
			<td>Andor Technologies</td>
			<td></td>
			<td>EMCCD camera with back-illuminated sensor</td>
		</tr>
		<tr>
			<td>LSM880 Airyscan</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>laser scanning confocal microscope with array detector</td>
		</tr>
		<tr>
			<td>Mitotracker CMXRos Red</td>
			<td>ThermoFischer</td>
			<td>M7512</td>
			<td>red live cell mitochondrial stain</td>
		</tr>
		<tr>
			<td>Mitotracker Deep Red FM</td>
			<td>ThermoFischer</td>
			<td>M22426</td>
			<td>far red live cell mitochondrial stain</td>
		</tr>
		<tr>
			<td>picoGreen</td>
			<td>ThermoFischer</td>
			<td>P7581</td>
			<td>cell permeant DNA stain</td>
		</tr>
		<tr>
			<td>Plan Apochromat 100x/1.46 Oil objective</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Gold</td>
			<td>ThermoFischer</td>
			<td>S11494</td>
			<td>cell permeant DNA stain</td>
		</tr>
		<tr>
			<td>Zen Black 2012 software</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>image acquisition and processing software</td>
		</tr>
	</tbody>
</table>

specific labeling of mitochondrial nucleoids for time-lapse structured illumination microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

Characterization of live cell labeling with SG 
First, the distribution of SG in the cells upon incubation with the dye at various dilutions was characterized by confocal microscopy. After incubation with high concentrations of SG or picoGreen, both dyes mostly labeled the nuclei and showed a punctate staining in the cytoplasm (Figure 1), similarly to published data for another positively charged cyanine dye (i.e., picoGreen)<sup class="...

Watch this Scientific Journal Video about Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy at JoVE.com

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

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

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.

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Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...