The authors thank Katherine Filpo Lopez for expert technical assistance. This work was supported by grants from the National Institutes of Health (NIH) (GM122589 and AG051047) to LP.
These studies used the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, funded in part through the NIH/NCI Cancer Center Support Grant P30CA013696.

Imaging of Mitochondrial Peroxide in Living Yeast Cells Using mtHyper7

The authors declare that they have no conflicts of interest.

In this protocol, a method is described for using mtHyPer7 as a biosensor to assess mitochondrial H2O2 in living&#160;budding yeast cells. The biosensor is constructed using a CRISPR-based method and introduced into a conserved gene-free region in the yeast genome without the use of selectable markers. Compared to plasmid-borne biosensors, integrated ones are expressed in all cells and at consistent levels, providing more reliable quantification results. No selectable markers are used to generate mtHyPer7-expressing cells, which allows for use of a wider range of strain backgrounds and facilitates the genetic modification of biosensor-expressing cells. The mtHyPer7 protein is correctly targeted to mitochondria without noticeable effects on mitochondrial morphology, function, distribution, or cellular growth rates. mtHyPer7 shows a dose-dependent response to externally added H2O2. Moreover, mtHyPer7 is able to report on the heterogeneity of mitochondrial quality with subcellular resolution. Finally, using a spinning-disk confocal microscope as opposed to wide-field microscopy for imaging mitochondrial-targeted biosensors causes less photobleaching to fluorophores and generates high-resolution images for analyzing subcellular differences.
Limitations and alternative approaches 
This method is not suitable for imaging cells for more than 10 min, as the cells will dry out under the coverslip. For longer-term imaging, it is better to use the agar pad method46 or to immobilize cells in a glass-bottom culture dish filled with SC medium.
The choice of biosensor should be guided by the concentration of the target under experimental conditions. If the sensitivity of HyPer7 is too high, a different HyPer version would be recommended, such as HyPer3 or HyPerRed47,48. However, it should be noted that other HyPer probes are more pH-sensitive. For higher sensitivity, the peroxiredoxin-based roGFP might be more appropriate (roGFP2Tsa2&#916;CR)27.
The oxidation steady state of the H2O2 sensor is tied to both oxidation and reduction rates. The oxidation rate of biosensors is mainly caused by H2O2, but the reduction rate depends on the antioxidant reduction systems active in the cell and organelle. It has been shown that HyPer7 is predominantly reduced by the thioredoxin system in yeast cytosol, and its reduction is faster than that of roGFP2Tsa2&#916;CR27. Therefore, the different reduction mechanisms and response dynamics of the probe should be taken into account when interpreting measurements of H2O2 biosensors. In particular, to infer H2O2 levels from the biosensor readout, it must be assumed that the reduction system maintains a constant capacity during the experiment. As an alternative to the scripts described here, a variety of other software has been made freely available for the analysis of redox sensors49.
Critical steps 
With any biosensor, it is critical to demonstrate that the biosensor itself does not affect the process being measured. Therefore, comparing the growth and mitochondrial morphology of strains under each experimental condition is important. Here, mitochondrial morphology is assessed using MitoTracker Red, which stains mitochondria in a membrane potential-dependent manner. However, comparison of the mitochondria in untransformed and biosensor-transformed cells can be accomplished by staining with tetramethylrhodamine methyl ester (TMRM), an alternative membrane potential-sensing mitochondrial vital dye, or MitoTracker Green, which stains mitochondria independent of membrane potential. If deleterious effects are suspected, reducing the expression level or changing the integration site may help.
Validating the dose-response behavior of the probe and the signal-to-noise ratio of the imaging technique are also essential for collecting robust results. If the variability within a group exceeds the variability between groups, differences become difficult to detect. Intragroup variability may result from true population variation, or from noise in the detection process. The key steps to increase the signal:noise ratio are image acquisition (pixel value range and noise), background subtraction, and thresholding.
Noise effects can also be reduced during the calculation steps. The most straightforward approach is to calculate the weighted mean intensity from the ratio image measurements (Results.csv), where each pixel represents the local ratio between the excitation efficiencies. This produces a &#34;pixelwise&#34; ratio. However, if the image signal:noise ratio is low, more robust results can be obtained by calculating the weighted mean intensity for an ROI in both the numerator and the denominator channels, and then calculating the ratio between these two weighted means (&#34;regionwise&#34; ratio).
To select a thresholding method, the Fiji command Image | Adjust | Auto Threshold can be used to automatically try all built-in Fiji methods. To evaluate segmentation (thresholding), a saved mask is converted into a selection by clicking on Edit | Selection | Create Selection, added to the ROI Manager (by pressing T), and then activated on the raw image file. If mitochondria are not adequately detected, a different segmentation method should be attempted.
When comparing images, it is essential to acquire all the images with identical imaging conditions, as well as to display all the images with identical contrast enhancement.
Mitochondrial movement must be considered when optimizing imaging conditions. If the mitochondria move significantly between excitation at 405 and 488 nm, the ratio image will not be accurate. It is recommended to keep the exposure time &#60;500 ms and to change excitation by the fastest method available (e.g. a trigger pulse or acousto-optic tunable filter). When capturing a Z stack, both excitations should be performed for each Z step before moving to the next Z step.
For display of the results, changes in hue (color) are more obvious to the human eye than changes in intensity. Therefore, the ratio value is converted to a color scale for easier visual interpretation. Colorized images can be unmodulated, where all the mitochondrial pixels appear at the same brightness, or intensity-modulated, where pixel intensity in the original image is used to set intensities in the colorized image.
Modification and troubleshooting 
As an alternative to confirming mitochondrial function by challenge with paraquat, cells may be replica-plated or inoculated into fermentable and non-fermentable carbon sources.
For background subtraction, rolling ball subtraction (by navigating to Process | Subtract Background&#8230;)&#160;may also be used to remove illumination nonuniformity. It should be ensured that the presence of cells does not alter the background being subtracted (by checking the option to Create Background and examining the result).
In summary, the mtHyPer7 probe provides a consistent, minimally invasive method to relate the morphological and functional status of yeast mitochondria in living cells, and allows the study of an important cellular stressor and signaling molecule in a genetically tractable, easily accessible model system.

Mitochondria are essential eukaryotic cellular organelles that are well known for their function in producing ATP through oxidative phosphorylation and electron transport1. In addition, mitochondria are sites for calcium storage, the synthesis of lipids, amino acids, fatty acid and iron-sulfur clusters, and signal transduction2,3. Within cells, mitochondria form a dynamic network with characteristic morphology and distribution, which varies according to cell type and metabolic state. Furthermore, although mitochondria can fuse and divide, not all the mitochondria in a cell are equivalent. Numerous studies have documented the functional heterogeneity of mitochondria within individual cells in attributes such as membrane potential and oxidative state4,5,6. This variation in mitochondrial function is due in part to damage to the organelle from mtDNA mutations (which occur at a higher rate than in nuclear DNA) and to oxidative damage by reactive oxygen species (ROS) generated both within and outside the organelle7,8,9. Damage to the organelle is mitigated by mitochondrial quality control mechanisms that repair the damage or eliminate mitochondria that are damaged beyond repair10.
Hydrogen peroxide (H2O2) is a reactive oxygen species that is a source of oxidative damage to cellular proteins, nucleic acids, and lipids. However, H2O2 also serves as a signaling molecule that regulates cellular activities through the reversible oxidation of thiols in target proteins11,12. H2O2 is produced from electrons that leak from the mitochondrial electron transport chain and by specific enzymes, such as NADPH oxidase and monoamine oxidases13,14,15,16,17,18,19,20. It is also inactivated by antioxidant systems, including those based on thioredoxin and glutathione21,22,23. Thus, analysis of mitochondrial H2O2 levels is critical for understanding the role of this metabolite in normal mitochondrial and cellular function and under conditions of oxidative stress.
The overall goal of this protocol is to detect mitochondrial H2O2 using a genetically encoded ratiometric H2O2 biosensor, HyPer7, that is targeted to the organelle (mtHyPer7). mtHyPer7 is a chimera consisting of the mitochondrial signal sequence from ATP9 (the Su9 presequence), a circularly permuted form of green fluorescent protein (GFP), and the H2O2-binding domain of the OxyR protein from Neisseria meningitidis24 (Figure 1). In circularly permuted GFP, the N- and C-termini of native GFP are fused and new termini are formed near the chromophore, which impart greater mobility to the protein and greater lability of its spectral characteristics compared to native GFP25. The interaction of mtHyPer7&#39;s OxyR domain with H2O2 is high-affinity, H2O2-selective, and leads to reversible oxidation of the conserved cysteine residues and disulfide bridge formation. Conformational changes associated with the oxidation of OxyR are transferred to the circularly permuted GFP in mtHyPer7, which results in a spectral shift in the excitation maximum of the mtHyPer7 chromophore from 405 nm in the reduced state to&#160;488 nm in the H2O2-oxidized state26. Thus, the ratio of fluorescence from mtHyPer7 in response to excitation at 488 nm versus 405 nm reflects the oxidation of the probe by H2O2.
Ideally, a biosensor should provide a real-time, absolute, quantitative readout of its target molecule. Unfortunately, however, this is not always possible in real-world measurements. In the case of oxidation sensors, such as mtHyPer7, the real-time readout is affected by the rate of reduction of the disulfide bridge. The reduction systems used by ROS biosensors differ, and these can dramatically alter probe response dynamics-as shown by the comparison of HyPer7, reduced by the thioredoxin system, and roGFP2-Tsa2&#916;CR, reduced by glutathione-in yeast cytosol27. Thus, to draw a conclusion about the relative H2O2 concentration from mtHyPer7 ratios, one must assume that the reduction system maintains a constant capacity during the experiment. Notwithstanding these considerations, HyPer7 and related probes have been used in various contexts to obtain information about H2O2 in living cells25,28,29.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65428/65428fig01.jpg" /> 
Figure 1: Design, molecular mechanism, and excitation/emission spectra of the H2O2 biosensor mtHyPer7. (A) The mtHyPer7 probe is derived by inserting circularly permuted GFP into the OxyR-RD domain from Neisseria meningitidis. It contains the mitochondrial targeting sequence from subunit 9 of the ATP synthase from Neurospora crassa (Su9). (B) Illustration of the H2O2-sensing mechanism of mtHyPer7. Oxidation of cysteines in the RD domain increases fluorescence emission upon excitation at 488 nm and decreases emission produced by excitation at 405 nm. (C) Excitation and emission spectra of HyPer7 in oxidized and reduced forms. This figure is reprinted with permission from Pak et al.24. Abbreviations: GFP = green fluorescent protein; cpGFP = circularly permuted GFP. <a href="https://www.jove.com/files/ftp_upload/65428/65428fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This ratiometric imaging of mtHyPer7 offers important benefits for mitochondrial H2O2 quantitation24,27; it provides an internal control for probe concentration. In addition, the shift in excitation peak produced by H2O2 exposure is not complete, even in saturating concentrations of H2O2. Thus, ratio imaging can increase the sensitivity by incorporating two spectral points in the analysis.
The mtHyPer7 probe used here has a very high affinity for H2O2 and relatively low sensitivity to pH24, and has been successfully targeted to Caenorhabditis elegans mitochondria30. This protein has also been used in yeast27,31. However, previous studies relied on plasmid-borne expression of mtHyPer7, which results in cell-to-cell variability in probe expression27. In addition, the construct described in this protocol was integrated into a conserved, gene-free region on chromosome X32 using a CRISPR-based approach for marker-free integration. Expression of the integrated biosensor gene is also controlled by the strong constitutive TEF1 promoter (Figure 2). As a result, there is more stable, consistent expression of the biosensor in yeast cell populations compared to that observed using plasmid-borne biosensor expression, and cells bearing the biosensor can be propagated without the need for selective media.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65428/65428fig02.jpg" /> 
Figure 2: Generation of mtHyPer7-expressing cells by CRISPR. The Cas9 and sgRNA-containing plasmid (YN2_1_LT58_X2site) and PCR-amplified mtHyPer7-containing biosensor construct are introduced into budding yeast cells by lithium acetate transformation. The gene-free insertion site on chromosome X (X2) is recognized and cut by Cas9 protein with the sgRNA, and the biosensor is integrated into the genome by homologous recombination. After identification of the successful transformants by microscopic screening, colony PCR, and sequencing, the Cas9 plasmid is removed (cured) by growth in non-selective media. Abbreviations: sgRNA = single guide RNA; TEF = transcription enhancer factor. <a href="https://www.jove.com/files/ftp_upload/65428/65428fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Finally, mtHyPer7 offers advantages over other ROS biosensors. For example, organic dyes used to detect ROS (e.g., dihydroethidium [DHE]2 and MitoSOX3) can produce uneven or nonspecific staining and are often delivered in solvents such as ethanol or dimethyl sulfoxide, which require additional controls for solvent effects. Another class of ROS biosensors are fluorescence resonance energy transfer (FRET)-based biosensors (e.g., Redoxfluor for cellular redox state4, and the peroxide sensors HSP-FRET5, OxyFRET6, and PerFRET6). These probes are genetically encoded and highly sensitive in principle and can be quantitatively targeted to mitochondria using well-characterized mitochondrial signal sequences. However, there are challenges in the use of FRET-based probes, including background due to cross-excitation and bleed-through, and stringent requirements for the proximity and orientation of the fluorophores for FRET to occur33,34. In addition, FRET probes consist of two fluorescent proteins that require larger constructs for expression in cells of interest compared to spectra-shifting probes. The protocol described here was developed to take advantage of the strengths of the HyPer7-based biosensor, and to use this compact, ratiometric, high-affinity, genetically encoded probe for quantitative imaging of peroxide in mitochondria in living yeast.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100x/1.45 Plan Apo Lambda objective lens</td>
			<td>Nikon</td>
			<td>MRD01905</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>#A9126</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD Difco</td>
			<td>#DF0145170</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>BD Difco</td>
			<td>#DF0118170</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Tryptone</td>
			<td>BD Difco</td>
			<td>#DF211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Yeast Extract</td>
			<td>BD Difco</td>
			<td>#DF0127179</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI</td>
			<td>New England Biolabs</td>
			<td>R0136S</td>
			<td></td>
		</tr>
		<tr>
			<td>BglII</td>
			<td>New England Biolabs</td>
			<td>R0144S</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicilin</td>
			<td>Sigma-Aldrich</td>
			<td>C1389</td>
			<td></td>
		</tr>
		<tr>
			<td>Carl Zeiss Immersol Immersion Oil</td>
			<td>Carl Zeiss</td>
			<td>444960</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose (D-(+)-Glucose)</td>
			<td>Sigma-Aldrich</td>
			<td>#G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>E. cloni 10G chemical competent cell</td>
			<td>Bioserch Technologies</td>
			<td>60108</td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td>NIH</td>
			<td></td>
			<td>Schindelin et al 2012</td>
		</tr>
		<tr>
			<td>G418 (Geneticin)</td>
			<td>Sigma-Aldrich</td>
			<td>A1720</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP emission filter</td>
			<td>Chroma</td>
			<td></td>
			<td>525/50</td>
		</tr>
		<tr>
			<td>Gibson assembly</td>
			<td>New England Biolabs</td>
			<td>E2611</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad Prism 7</td>
			<td>GraphPad</td>
			<td></td>
			<td>https://www.graphpad.com/scientific-software/prism/</td>
		</tr>
		<tr>
			<td>H2O2 (stable)</td>
			<td>Sigma-Aldrich</td>
			<td>H1009</td>
			<td></td>
		</tr>
		<tr>
			<td>HO-pGPD-mito-roGFP-KanMX6-HO</td>
			<td>Pon Lab</td>
			<td>JYE057/EP41</td>
			<td>Liao et al 20201</td>
		</tr>
		<tr>
			<td>Incubator Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>E24</td>
			<td></td>
		</tr>
		<tr>
			<td>KAPA HiFi PCR kit</td>
			<td>Roche Sequencing and Life Science, Kapa Biosystems, Wilmington, MA</td>
			<td>KK1006</td>
			<td></td>
		</tr>
		<tr>
			<td>L-arginine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>#A8094</td>
			<td></td>
		</tr>
		<tr>
			<td>laser</td>
			<td>Agilent</td>
			<td></td>
			<td>405 and 488 nm</td>
		</tr>
		<tr>
			<td>L-histidine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>#H5659</td>
			<td></td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>Sigma-Aldrich</td>
			<td>#L8912</td>
			<td></td>
		</tr>
		<tr>
			<td>L-lysine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>#L8662</td>
			<td></td>
		</tr>
		<tr>
			<td>L-methionine</td>
			<td>Sigma-Aldrich</td>
			<td>#M9625</td>
			<td></td>
		</tr>
		<tr>
			<td>L-phenylalanine</td>
			<td>Sigma-Aldrich</td>
			<td>#P5482</td>
			<td></td>
		</tr>
		<tr>
			<td>L-tryptophan</td>
			<td>Sigma-Aldrich</td>
			<td>#T8941</td>
			<td></td>
		</tr>
		<tr>
			<td>L-tyrosine</td>
			<td>Sigma-Aldrich</td>
			<td>#T8566</td>
			<td></td>
		</tr>
		<tr>
			<td>mHyPer7 plasmid</td>
			<td>This study</td>
			<td>JYE116</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope coverslips</td>
			<td>ThermoScientific</td>
			<td>3406</td>
			<td>#1.5 (170 &#181;m thickness)</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>ThermoScientific</td>
			<td>3050</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Red CM-H2Xros</td>
			<td>ThermoFisherScientific</td>
			<td>M7513</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuilder HiFi Assembly Master Mix</td>
			<td>New England Biolabs</td>
			<td>E2621</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscope acquisition software</td>
		</tr>
		<tr>
			<td>Nikon Ti Eclipse inverted microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraquat (Methyl viologen dichloride hydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>Cat. #856177&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>RStudio</td>
			<td>Posit.co</td>
			<td>Free desktop version</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Beckman</td>
			<td>BU530</td>
			<td></td>
		</tr>
		<tr>
			<td>Stagetop incubator</td>
			<td>Tokai Hit</td>
			<td>INU</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Sigma-Aldrich</td>
			<td>#U1128</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast nitrogen base (YNB) containing ammonium sulfate without amino acids</td>
			<td>BD Difco</td>
			<td>#DF0919073</td>
			<td></td>
		</tr>
		<tr>
			<td>YN2_1_LT58_X2site</td>
			<td>Addgene</td>
			<td>177705</td>
			<td>Pianale et al 2021</td>
		</tr>
		<tr>
			<td>Zyla 4.2 sCMOS camera</td>
			<td>Andor</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging of mthyper7, a ratiometric biosensor for mitochondrial peroxide, in living yeast cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

To confirm that mtHyPer7 is a suitable probe to assess mitochondrial H2O2, the localization of mtHyPer7 and its effect on yeast mitochondria and cell health were assessed. To assess the targeting of mtHyPer7, mitochondria were stained with the vital mitochondria-specific dye MitoTracker Red in control yeast cells and yeast that express mtHyPer7. Using MitoTracker Red staining, mitochondria were resolved as long tubular structures that aligned along the mother-bud axis and accumulated in the tip of the bud and the tip of the mother cell distal to the bud41. Mitochondrial morphology was similar in the control and mtHyPer7-expressing cells. In addition, mtHyPer7 co-localized with MitoTracker Red-stained mitochondria (Figure 3A). Thus, mtHyPer7 was efficiently and quantitatively targeted to mitochondria without affecting normal mitochondrial morphology or distribution.
Next, additional validation experiments were performed to assess the effect of mtHyPer7 on cellular fitness and sensitivity to oxidative stress in mitochondria. The growth rates of mtHyPer7-expressing cells in rich or synthetic glucose-based media (YPD or SC, respectively) are similar to those of untransformed wild-type cells (Figure 3B,D). To assess the possible effects on oxidative stress in mitochondria, control and mtHyPer7-expressing yeast were treated with low levels of paraquat, a redox-active small molecule that accumulates in mitochondria and results in elevated superoxide levels in the organelle24,27. If mtHyPer7 expression either induces oxidative stress in mitochondria or protects mitochondria from oxidative stress, then mtHyPer7-expressing cells should exhibit an increased or decreased sensitivity to paraquat treatment, respectively. Treatment with low levels of paraquat resulted in a decrease in yeast growth rates. Moreover, the growth rates of wild-type and mtHyPer7-expressing cells in the presence of paraquat were similar (Figure 3C,E). Therefore, expression of the biosensor did not create significant cellular stresses in budding yeast cells or alter the sensitivity of yeast to mitochondrial oxidative stress.
Given the evidence that mtHyPer7 is suitable for studying mitochondrial H2O2 in budding yeast, it was tested whether mtHyPer7 could detect mitochondrial&#160;H2O2 in mid-log phase wild-type yeast and changes in mitochondrial H2O2 induced by externally added H2O2. An H2O2 titration experiment was performed and the mean oxidized:reduced (O/R) mtHyPer7 ratio was measured in mitochondria.
The automated analysis scripts produced several output files.
The ratio image (ratio.tif): A Z stack consisting of the ratio of the background-corrected, thresholded 488 nm and 405 nm stacks. This stack can be viewed in Fiji without additional processing; however, enhancing the contrast and/or colorizing the image as described in protocol step 4.3 or 5.6 prior to viewing is recommended.
The mask image (mask.tif): A Z stack consisting of the thresholded mitochondrial areas used for analysis. This image must be used to evaluate the accuracy of the thresholding.
The ROIs used for analysis (ROIs.zip): When this file is opened in Fiji, along with the source image, the ROIs are superimposed on the image to cross-reference the results table and source image. Each ROI is renamed with a cell number and ROI number.
Measurement tables (NumResults.csv, DenomResults.csv, Results.csv) containing the area, mean, and integrated density of thresholded mitochondria for each slice in the numerator, denominator, and ratio images, respectively. In slices where mitochondria were absent or out of focus, NaN is recorded.
A log file (Log.txt) documenting the options used for background and noise correction, thresholding, and ratio calculation.
The O/R ratio of mtHyPer7 showed a dose-dependent response to H2O2 concentration, which reached a plateau at 1-2 mM externally added H2O2 (Figure 7). Surprisingly, the HyPer7 ratio was lower in the cells exposed to 0.1 mM H2O2 than in the control cells, although this difference was not statistically significant. One explanation for this phenomenon may be a hormesis response, in which exposure to a low level of stressor may induce stress responses, such as antioxidant mechanisms, which in turn lower the amount of ROS detectable by the probe. Higher levels of stressors, in contrast, may overwhelm the internal stress responses and result in a higher readout from HyPer7.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65428/65428fig07.jpg" /> 
Figure 7: Response of mtHyPer7 to externally added H2O2. (A)&#160;Maximum projection of 405 nm and 488 nm excited images and average intensity projection of ratiometric images of mtHyPer7 in mid-log phase cells exposed to different concentrations of H2O2. Pseudocolor indicates the oxidized:reduced mtHyPer7 ratio (scale at bottom). Scale bar = 1 &#181;m. The cell outline is shown in white; n &#62; 100 cells per condition. (B)&#160;Quantification of the mtHyPer7 oxidized:reduced ratio in budding yeast cells treated with different concentrations of H2O2. The means of five independent trials are shown, with differently shaped and colored symbols for each trial. Mean &#177; SEM of the ratio of oxidized:reduced mtHyPer7: 1.794 &#177; 0.07627 (no treatment), 1.357 &#177; 0.03295 (0.1 mM), 2.571 &#177; 0.3186 (0.5 mM), 6.693 &#177; 0.5194 (1 mM), 7.017 &#177; 0.1197 (2 mM). p &#60; 0.0001 (one-way ANOVA with Tukey&#39;s multiple comparisons test). p values are denoted as: ****p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/65428/65428fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Finally, previous studies revealed that fitter mitochondria, which are more reduced and contain less superoxide, are preferentially inherited by yeast daughter cells4, and that cytosolic catalases are transported to and activated in yeast daughter cells42,43,44,45. These studies document the asymmetric inheritance of mitochondria during yeast cell division and a role for this process in daughter cell fitness and lifespan, as well as mother-daughter age asymmetry. To test whether differences in H2O2 exist between mothers and buds, mtHyPer7 was measured in buds and mother cells. Differences in mitochondrial H2O2 were observed within yeast cells, and a subtle but statistically significant decrease in H2O2 biosensor readout was detected in mitochondria in the bud compared to those in the mother cell (Figure 8). These findings are consistent with previous findings that mitochondria in the bud are better protected from oxidative stress. They also provide documentation that mtHyPer7 can provide a quantitative readout for mitochondrial H2O2 with cellular and subcellular resolution in budding yeast.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/65428/65428fig08.jpg" /> 
Figure 8: Mitochondrial H2O2 level is lower in the daughter cell. (A)&#160;Maximum projection of a ratiometric image of mtHyPer7 in a representative cell. Pseudocolor indicates the oxidized:reduced mtHyPer7 ratio (scale at bottom). Subcellular differences in the ratio are evident (arrowheads). Scale bar = 1 &#181;m. Cell outline: white.&#160;(B) Difference between the mtHyPer7 ratio in the bud and mother cell. For each individual cell, the mother ratio value was subtracted from the bud ratio value and plotted as a point. n = 193 cells pooled from five independent experiments, shown with differently colored symbols for each experiment. Mean &#177; SEM of the difference between the mtHyPer7 ratio in the bud and mother cells: -0.04297 &#177; 0.01266. p = 0.0008 (paired t-test) <a href="https://www.jove.com/files/ftp_upload/65428/65428fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Table S1: Strains used in this study. List of yeast strains used. <a href="https://www.jove.com/files/ftp_upload/65428/65428_Supplemental Table 1 Strains used in this study-R2.xls" target="_blank">Please click here to download this File.</a>
Supplemental Table S2: Plasmids used in this study. List of plasmids used. <a href="https://www.jove.com/files/ftp_upload/65428/Supplemental Table 2 Plasmids used in this study-R2.xls" target="_blank">Please click here to download this File.</a>
Supplemental Table S3: Primers used in this study. List of primers used. <a href="https://www.jove.com/files/ftp_upload/65428/65428_Supplemental Table 3 Primers used in this study-R2.xls" target="_blank">Please click here to download this File.</a>
Supplemental File 1: Protocol for biosensor plasmid construction. <a href="https://www.jove.com/files/ftp_upload/65428/65428_Yang et al Supp Protocol 2023.docx" target="_blank">Please click here to download this File.</a>
Supplemental File 2:&#160;Scripts for automated analysis. For each script, sample input files and output files are supplied. <a href="https://www.jove.com/files/ftp_upload/65428/biosensor-supplemental-coding-files.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Advancing Mitochondrial Research Â– mtHyper7 Biosensor for Subcellular Analysis at JoVE.com

Author Spotlight: Advancing Mitochondrial Research - mtHyper7 Biosensor for Subcellular Analysis 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...

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2.0K Views.  Columbia University Irving Medical Center. Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

Video: Author Spotlight: Advancing Mitochondrial Research - mtHyper7 Biosensor for Subcellular Analysis 

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

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

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

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.

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 ...