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.

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

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

Mitochondria are organelles within cells that facilitate energy mobilization, central metabolism, production of vital macromolecules, calcium storage, and signal transduction. As a result, they play a crucial role in cellular function, fitness, and lifespan control. Our research focuses on investigating the impact of mitochondria on aging using the budding yeast Saccharomyces cerevisiae as a model organism.Live cell imaging is our best tool to observe variation in mitochondrial function. Here we describe a method that utilizes a genetically encoded ratio metric spectral shifting biosensor called mitochondrial HyPer7. mtHyPer7 detects hydrogen peroxide, a reactive oxygen species, that can damage cellular constituents, but also functions as a signaling molecule in mitochondria of live cells.mtHyPer7 has a high affinity for hydrogen peroxide and low sensitivity to pH. It is integrated into the yeast genome, which results in stable and consistent expression in yeast cell populations. Finally, it is quantitatively targeted to mitochondria and has no detectable effect on cellular or mitochondrial function or fitness.Biosensors like mtHyPer7 are critical for studying mitochondrial function at subcellular resolution, for finding mechanisms underlying mitochondrial quality control, and for examining how that process affects cell fitness, feed, and lifespan. The mtHyPer7 stain provides consistent laboring in contrast to organic dyes utilized for reactive oxygen species detection. Compared to FRET-based sensors, mtHyPer7 is more compact and avoids issues such as cross-excitation and bleed through, as well as the precise positioning and the orientation requirements for FRET.

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  mtHyper7 Biosensor for Subcellular Analysis at JoVE.com

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.

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

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Research

JoVE Journal

Biology

Preparation of Yeast Cells for Imaging, Optimization of Imaging Conditions, and Image Collection

To begin, take one milliliter of a mid-log phase budding yeast cell culture, expressing mitochondria-targeted HyPer7. Centrifuge the cells at 6, 000 G for 30 seconds, and remove the supernatant, leaving 10 to 20 microliters in the tube. Re-suspend the cell pellet by gently mixing it with residual media using a micro-pipette.Next, use an air blower or lint-free tissue to clean a glass microscope slide, and add 1.8 microliters of the cell suspension to the slide. Finally, cover the cells with number 1.5 glass cover slip by lowering it slowly at an angle to avoid introducing bubbles. Then place the slide on the microscope stage.To image the cells, set up the acquisition conditions to ensure a detectable signal and acceptable resolution in each fluorescence channel. For example, on a spinning disc confocal microscope with an sCMOS camera use 2x2 binning, 20 to 40%laser power, and 200 to 600 milliseconds exposure. Next, examine the image histogram.In a 12-bit image ensure the total dynamic range of the pixel values is at least several hundred gray levels without saturation. Additionally, ensure the range is one order of magnitude higher than the noise level. To calculate the noise level, measure the standard deviation of pixel values in a cell-free area of an image.Then collect time-lapse images with no delay between acquisitions to evaluate the effects of the imaging conditions on signal stability and oxidative stress in mitochondria. Using the microscope acquisition software or ImageJ, measure the average pixel value in each fluorescence channel to ensure signal stability. If the experiment does not involve time-lapse imaging, ensure that the fluorescence is stable over two or three repeated Z-stacks.Acquire images with a Z interval of 0.5 micrometers in a total stack depth of six micrometers for budding yeast. If collecting Z-stacks ensure that the Z interval is the same for all images in the dataset and include the entire cell. Also acquire transmitted light images to document cell boundaries.

Semiautomated Analysis of mtHyPer7 in Yeast Cells with Scripts 

To begin, open a multichannel Z stack image in Fiji, and open the desired biosensor analysis script file. Click Run in the script editor window to run the script. Then enter the requested information in the dialogue window that appears.Select the channel numbers of the numerator and denominator for ratio calculation. For mitochondria targeted HyPer7, the numerator is the channel excited at 488 nanometers and the denominator is the channel excited at 405 nanometers. Select the channel number of the transmitted light image if present, or zero if there is none.Then choose the desired background subtraction method. If the background is uniform, click select an image area to measure the background level in the image. Afterward, choose the desired noise subtraction method to reduce random variation in the detector readout.Click Select an image area. Select the fixed value option to enter a previously measured noise level, which usually works well if the imaging conditions are kept constant. For accurate and consistent detection of mitochondria, select a thresholding algorithm such as Otsu or Maximum Entropy.Use the same algorithm for all images in an experiment, but ensure that mitochondria are accurately recognized. Then select the number of regions of interest per cell. For example, if measuring mother bud differences, select two.Select the output folder where measurements and ratio images will be saved. For background or noise correction, choose Select an image area. Follow the prompts to draw a background area using the rectangle ROI tool and click Okay.While analyzing individual cells or subcellular regions, draw regions of interest based on the brightfield image. The ROI does not need to precisely match the cell outline as only thresholded mitochondria within the ROI will be measured. After creating each ROI, press T to add the selected ROI to the ROI Manager.Check Show All in the ROI Manager to document the marked cells. Each added region will appear as a numbered item in the ROI Manager list. If analyzing more than one ROI per cell, mark the ROIs in the same order for each analyzed cell.After all the desired ROIs have been added to the ROI manager, click Okay in the Mark Cells dialogue window. Next, select the measurement table format. In the Multi Measure dialogue window, check measure-all-slices and one-row-per-slice options to produce a table with a desired format.Use the process multi ROI tables. rscript to process the tables created with the one-row-per-slice option. Do not check the append-results option.Save the output files to the selected folder using the script. Now open the ratio Z stack image in Fiji to generate a colorized ratio image. Then open the colorize_ratio_image.ijm script file and click Run in the script editor window. A dialogue window prompting to enter the requested information will appear. In the unmodulated option, all mitochondrial pixels appear at the same brightness.Some images may appear noisy as dim and bright pixels contribute to the ratio image. Use the intensity modulated option to reduce the noise. In the minimum and maximum displayed values method, choose values near the average minimum and maximum values observed in an experiment.To ensure consistency, acquire all images using the same imaging conditions and display all images using the same minimum and maximum values. Then select the projection mode to project the Z stack and show the entire mitochondrial population before colorization. For a maximum intensity projection, select maximum.To create an average intensity projection, select average. Finally, select the folder to save the colorized images. If the unmodulated option is selected, choose a color scheme in the dialogue window that appears.Utilize the fire or rainbow RGB lookup tables built into Fiji or any desired lookup table in ImageJ's LUT format. Use the script to save the output files to the selected folder. The oxidized reduced ratio of mitochondria tagged HyPer7 showed a dose dependent response to hydrogen peroxide concentration, which reached a plateau at one to two millimolar externally added hydrogen peroxide.Differences in mitochondrial hydrogen peroxide were observed within the yeast cells. A statistically significant decrease in hydrogen peroxide biosensor readout was detected in mitochondria in the bud compared to the mother cell.