K. Wong, S.T.C. Wong are supported by the funding from Functional and Molecular Imaging Center, Brigham and Women's hospital 
K. Wong, S.T.C. Wong are supported by the Center for Biotechnology and Informatics, The Methodist Hospital Research Institute, Weill Cornell Medical College. 
K.K. Kwong, I. Chen, J-Q Ren are supported by the funding from the Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital. 
Lenuta Kloetzer, Braden Kuo are supported by funding from the Neuroenteric Research Center, Massachusetts General Hospital 
The authors thank Drs. Q. Zeng and X. Xu of Optical Imaging Lab, Brigham and Women&#x2019;s Hospital for technical help.

<ol>
 <li>Female HO-1-luciferase transgenic mice (age of 4-6 weeks) can be purchased from Taconic Farms, Inc. Upon arrival, mice are placed in an animal housing facility for at least 4 days to allow accommodation. </li>
 <li>Preparation for bioluminescence imaging: 
 <ol style="list-style-type:lower-alpha;">
 <li>Weigh each HO-1 mouse. Animal weight is required for calculating the dosage of luciferin. </li>
 <li>Hair removal: Use a cotton swap to apply hair remover Nair&reg; over the abdomen and/or back of the animal. Waiting for 5 to 10 seconds. Use a clean cotton swap to wipe off hair. Dip a piece of tissue in distilled water to wipe clean remaining hair.</li>
 <li>Place a non-fluorescent black paper (Strathmore Artagain&reg; black paper) on the imaging platform of an IVIS 100 station to reduce background noise. </li>
 <li>Prepare luciferin (from Xenogen Corporation) solution at concentration of 7.5 mg/ml (dissolved in sterile H2O). The dose of luciferin is 65.5 mg/kg body weight. Thus, a 20 gram mouse requires intraperitoneal injection of 0.175 ml luciferin solution. To achieve this, a single injection volume of luciferin solution (0.175ml) is preloaded to a syringe with a 26-gauge needle </li>
 </ol></li>
 <li>Gua Sha procedure: Gua Sha is applied only once before running the bioluminescence imaging protocol. Since Gua Sha is not painful, the mouse needs only to be briefly anesthetized by isoflurane to stay calm. Gua Sha procedure includes the following steps:
 <ol style="list-style-type:lower-alpha;">
 <li>Apply cooking oil or distilled water to lubricate skin areas targeted by Gua Sha a few times during the Gua Sha procedure. </li>
 <li>Repetitively scrape the hair-free region of the back of the mouse in gentle but firm force using a ceramic soup spoon or a plastic spoon.</li>
 <li>The scraping continues until the back skin turns red, which is a sign of subcutaneous blood extravasation, usually achieved within 2 to 3 minutes. </li>
 </ol></li>
 <li>Bioluminescence imaging may be started immediately or several hours after Gua Sha as HO-1 upregulation is built up slowly over a few hours. The procedure for bioluminescence imaging is:
 <ol style="list-style-type:lower-alpha;">
 <li>Anesthetize the mouse in an anesthesia induction chamber filled with a mixture of isoflurane (1.5%) and medical grade air.</li>
	<li>Once the mouse is anesthetized, move the mouse to the imaging chamber on the IVIS 100 optical imaging station. Position the mouse in a supine position (abdomen up). The imaging chamber is continuously infused with 1.5% of isoflurane. The imaging platform is heated at 37&deg;C to keep the mouse warm.</li>
	<li>Set the imaging acquisition at "medium binning" and set exposure time to be 30 seconds. Start to acquire images. Set the machine to repeat the imaging acquisition every three minutes, either manually or automatically. After the acquisition of the first image, a region of interest (ROI) is drawn to cover the abdominal, chest, and head area. This ROI is then copied and pasted to the following images using "Living Image&reg;", software accompanying the IVIS 100 station.</li>
	<li>Signal intensity is measured in photons per second. Mark time for the signal intensity to reach its peak value and keep on imaging the mouse for about 5 to 10 minutes after the peak time.</li>
	<li>When the imaging acquisition at the supine position is done, flip the mouse over and lay it in the prone position (back up). Continue imaging the animal for 5 to 10 minutes with the ROI now drawn to cover the back and the head area in the use of the software "Living Image&reg;". The choice of imaging the prone position after the supine position is arbitrary. One can choose to start with the prone position, depending on the primary organs of interest. Another option is to image the supine and prone positions at separate experiments.</li>
	<li>When the imaging acquisition at the prone position is finished, turn off isoflurane and move mouse from the imaging chamber to the induction chamber to recover. The induction chamber is now infused with medical air only and the mouse usually wakes up in less than one minute.</li>
	<li>Save imaging data for post-processing.</li>
	<li>After the initial Imaging study, additional imaging acquisitions are carried out for several days after Gua Sha. One sample schedule is repeated imaging at the 12th hour, 24th hour, 36th hour, 48th hour, 72nd hour and 120th hour but the exact timing of the imaging schedule is flexible. Additional imaging sessions may be added if a longer follow-up is needed. Before imaging, repeat the hair removing procedure if the hair has grown back.</li>
 </ol>
 </li>
 <li>For the control group, repeat all procedure but skip Gua Sha (step 3). If the same group of mice is used for both control and Gua Sha, the control experiment should be carried out preferably before the Gua sha procedure or at least one month afterwards.</li>
</ol>
Representative Results:
The bioluminescence images in figure 1 show in vivo upregulation of HO-1 in response to Gua Sha. The graph in Figure 2 shows the quantitative temporal change over 120 hours in optical flux (photons/sec) from the whole body of the same mouse related to Gua Sha...
<img src="/files/ftp_upload/1385/figure1tmb.jpg" alt="figure 1" border="0" /> Figure 1. From left to right, representative images of the front view (supine) of same mouse before Gua Sha, at 18 hours, 36 hours and 120 hours post Gua Sha, respectively. After Gua Sha, one observes the progress of significant signal intensity increase in multiple organs which encompass regions of the gastrointestinal tract, the genital tract, the liver, kidneys (from the back view, not shown), and others. Please <a href="https://www.jove.com/files/ftp_upload/1385/figure1.jpg">click here</a> for a larger version of figure 1.
<img src="/files/ftp_upload/1385/figure2tmb.jpg" alt="figure 2" border="0" /> Figure 2. Quantitative change of flux (photons/sec) from the whole body tracked over 120 hours following Gua Sha in the same mouse of Figure 1. Please <a href="https://www.jove.com/files/ftp_upload/1385/figure2.jpg">click here</a> to see a larger version of figure 2.

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The authors have nothing to disclose.

Transcription of HO-1, an inducible form of heme oxygenase, is upregulated by many factors including heme, hydrogen peroxide, UV irradiation, hypoxia, and physical stresses. Whole body imaging such as the reported HO-1 bioluminescence protocol provides a quick snapshot of systemic HO-1 expression in multiple organs. In small animals, bioluminescence imaging allows high-sensitivity in-vivo real-time quantitative longitudinal optical assessment of alterations in systemic gene expression, as shown. Bioluminescence molecul...

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bioluminescence imaging of heme oxygenase-1 upregulation in the gua sha procedure

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol for bioluminescent optical imaging of HO-1-luciferase transgenic mice reported in this manuscript provides a rapid in vivo assay of the upregulation of the heme oxygenase-1 (HO-1) gene expression in response to the Gua Sha procedure. HO-1 has long been known to provide cytoprotection against oxidative stress. The upregulation of HO-1, assessed by the bioluminescence output, is thought to represent an antioxidative response to circulating hemoglobin products released by Gua Sha. Gua Sha was administered by repeated strokes of a smooth spoon edge over lubricated skin on the back or other targeted body part of the transgenic mouse until petechiae (splinter hemorrhages) or ecchymosis (bruises) indicative of extravasation of blood from subcutaneous capillaries was observed. After Gua Sha, bioluminescence imaging sessions were carried out daily for several days to follow the dynamics of HO-1 expression in multiple internal organs.

Watch this Scientific Journal Video about Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure at JoVE.com

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

bioluminescence-imaging-heme-oxygenase-1-upregulation-gua-sha

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JoVE Journal

Biology

14.0K Aufrufe.  Massachusetts General Hospital, Harvard Medical School. Gua Sha, traditionelle chinesische therapeutische Hautgeschabsel, verursacht subkutane mikrovaskuläre Blutextravasat. Wir berichten über einen Protokoll der Biolumineszenz-Bildgebung von HO-1- Luciferase Transgenen Mäusen zeigen, dass Gua Sha Häm hochreguliert Oxygenase-1 (HO-1) in mehreren Organen.

Serie: Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

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 ...

Imaging of Mitochondrial Peroxide in Living Yeast Cells Using mtHyper7

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 

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, ...