Mitochondrial superoxide flashes (mSOFs) are stochastic events of quantal mitochondrial superoxide generation. Here, we used flexor digitorum brevis muscle fibers from transgenic mice with muscle-specific expression of a novel mitochondrial-targeted superoxide biosensor (mt-cpYFP) to characterize mSOF activity in skeletal muscle at rest, following intense activity, and under pathological conditions. Results demonstrate that mSOF activity in muscle depended on electron transport chain and adenine nucleotide translocase functionality, but it was independent of cyclophilin-D-mediated mitochondrial permeability transition pore activity. The diverse spatial dimensions of individual mSOF events were found to reflect a complex underlying morphology of the mitochondrial network, as examined by electron microscopy. Muscle activity regulated mSOF activity in a biphasic manner. Specifically, mSOF frequency was significantly increased following brief tetanic stimulation (18.1 ± 1.6 to 22.3 ± 2.0 flashes/1000 μm²·100 s before and after 5 tetani) and markedly decreased (to 7.7 ± 1.6 flashes/1000 μm²·100 s) following prolonged tetanic stimulation (40 tetani). A significant temperature-dependent increase in mSOF frequency (11.9 ± 0.8 and 19.8 ± 2.6 flashes/1000 μm²·100 s at 23°C and 37°C) was observed in fibers from RYR1(Y522S/WT) mice, a mouse model of malignant hyperthermia and heat-induced hypermetabolism. Together, these results demonstrate that mSOF activity is a highly sensitive biomarker of mitochondrial respiration and the cellular metabolic state of muscle during physiological activity and pathological oxidative stress

A 40 × 35 × 25-mm3 specimen of human breast consisting mostly of fat and connective tissue was imaged using a 3-T magnetic resonance scanner. The resolutions in the image plane and in the orthogonal direction were 130 μm and 150 μm, respectively. Initial processing to prepare the data for segmentation consisted of contrast inversion, interpolation, and noise reduction. Noise reduction used a multilevel bidirectional median filter to preserve edges. The volume of data was segmented into regions of fat and connective tissue by using a combination of local and global thresholding. Local thresholding was performed to preserve fine detail, while global thresholding was performed to minimize the interclass variance between voxels classified as background and voxels classified as object. After smoothing the data to avoid aliasing artifacts, the segmented data volume was visualized using iso-surfaces. The isosurfaces were enhanced using transparency, lighting, shading, reflectance, and animation. Computations of pulse propagation through the model illustrate its utility for the study of ultrasound aberration. The results show the feasibility of using the described combination of methods to demonstrate tissue morphology in a form that provides insight about the way ultrasound beams are aberrated in three dimensions by tissue.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg 1 . We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 μm. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke. Video LinkThe video component of this article can be found at https://www.jove.com/video/3466/ Protocol Preparing the animal for imagingNote: We use adult c57BL mice. Different transgenic strains can be used depending on the research question. Microvascular surgeries and pulse oximetry are facilitated in mice with white fur (e.g. FVB strain).1. Prepare the surgical site at the bench. All surgical instruments need to be autoclaved or disinfected with 70% ethanol. 2. Insert a rectal probe, and continuously measure the animal's body temperature throughout the procedure 3. Anesthetize the mouse using initially 5% isoflurane. In 15-30 s, when the animal is not moving, place it on a heating pad (37 °C), and apply a face mask for the continuous delivery of air containing 1.3-1.5% isoflurane). Make sure that the animal is not responding to a pain stimulus (e.g. tail pinch). 4. Use artificial tear gel to cover the mouse's eyes, to prevent exposure to dry air. 5. Using an electric razor, remove hair from the head and from both thighs. Apply hair remover (e.g. Nair) to the thigh for 2 min, and then wipe it carefully to remove the remaining hair. This thigh will be used for oximetry. 6. Disinfect the scalp using a 10% povidone-iodine solution and 70% ethanol. Preparing the open skull cranial window1. Starting 5 mm caudal to the skull, make an incision in the scalp with scissors, and advance one centimeter. Move skin to the sides to expose the skull. 2. To remove the membranes on top of the skull apply 10% ferric chloride solution to the top of the skull. Wipe...

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