Transmission Electron Microscopy for Analysis of Mitochondria in Mouse Skeletal Muscle

McMillan, Joseph D; Eisenback, Michael A.

doi:10.21769/bioprotoc.2455

Cited by 12 publications

(9 citation statements)

References 16 publications

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“…Super resolution microscopy techniques, such as stimulated emission depletion, have improved the resolution of optical microscopy to less than 50 nm, and are used to reveal the distribution of proteins involved in activation at neuromuscular junctions in rats (Nishimune, Badawi, Mori, & Shigemoto, 2016). Transmission electron microscopy is a powerful technique for ultrastructural studies, and is useful in studying the cell orgenelles' structure such as the mitochondria of skeletal muscle (McMillan & Eisenback, 2018). Therefore, it is necessary to analyze the muscles in multiple ways by combining these technologies.…”

Section: Discussionmentioning

confidence: 99%

“…The skeletal muscle is the most abundant tissue in the human body and it regulates various functions, including movement and whole‐body metabolism. It is a highly plastic tissue that shows morphological and metabolic changes in response to a number of extracellular signals (McMillan & Eisenback, 2018). It accounts for approximately 40% of the total body weight, 50–75% of whole‐body proteins, and 30–50% of whole‐body protein turnover.…”

Section: Introductionmentioning

confidence: 99%

“…Animal models are widely used to study the structure at the level of cell organelles, such as muscle mitochondria, microvascular structure, special arrangement of muscle fibers, and mechanism of force generation in muscles (McMillan & Eisenback, 2018; Powers, Nishikawa, Joumaa, & Herzog, 2016; Schaad et al, 2017). Animals' soleus muscles are also widely used in muscle studies.…”

Section: Introductionmentioning

confidence: 99%

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Micro‐ and nano‐tomography analysis of mouse soleus muscle using radiation

Jang

Lee

2021

Microscopy Res & Technique

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In this study, we analyze radiation images of muscle structure of mice soleus muscles using radiation source‐based microtomography and nanotomography. Soleus muscle samples were collected for analysis from 8‐week‐old male Institute of Cancer Research mice. First, phase‐contrast X‐ray microtomography was employed in these experiments. Then to obtain images with excellent contrast, imaging was performed using monochromatic light with excellent transmission power. To analyze additional muscle structures in higher magnification images than these images, nanotomography was performed, which facilitated obtaining high‐magnification and high‐resolution images. Muscle tissue microstructures were confirmed through three‐dimensional images obtained from phase‐contrast X‐ray microtomography. Thus, the muscle tissue's overall shape at microscopic level can be captured. Additionally, a single muscle fiber was examined using hard X‐ray nano‐imaging, through which we could observe the alignment of countless myofibrils, that is, actin and myosin filaments in the muscle fibers. Thus, the methodology adopted here proved to be advantageous in analyzing the muscle tissue's overall structure with microtomography and in observing the myofibrils in detail using nanotomography.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Micro‐ and nano‐tomography analysis of mouse soleus muscle using radiation

Jang

Lee

2021

Microscopy Res & Technique

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“…The samples were absorbed on a TEM copper grid, and then the electron micrograph was recorded by 120kV Tecnai T12 transmission electron microscope (FEI). For liver tissue, the DAB and H2O2 steps were removed and further examined under transmission electron microscopy using standard operating procedures 30 , 31 .…”

Section: Transmission Electron Microscopy Imagingmentioning

confidence: 99%

LBP resists hepatic oxidative stress by regulating LD homeostasis

Fang

Zhang

Shen

et al. 2023

Preprint

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Lipopolysaccharide-binding protein (LBP), an acute-phase protein, is expressed in inflammatory response and stress1,2, but its intracellular function is still unclear. Here, we uncover the profound paradigm of LBP as an antioxidant by coupling lipid droplet (LD) metabolism and redox signal, which protects hepatocytes from lipid peroxidation. LBP is upregulated and identified in LDs of hepatocytes after 24h fasting. Hepatic knock-in of LBP causes severe steatosis and LD accumulation on a high-fat diet. We showed that the C-segment groove of LBP conferred triglyceride (TG) capture activity, resulting in the LD-translocation of LBP and increasing the LD stability. Thus, a combination of LBP and unsaturated TG prevents lipid peroxidation under oxidative stress, whereas LBP was shuttled-out from LD after N-Acetyl-L-cysteine (NAC) treatment to promote phospholipid synthesis and lipolysis. Moreover, we found that LBP as a redox regulator of PRDX4, can sense the oxidative stress of cells and achieve precise control of LDs homeostasis. Stresses, like chronic jet lag and forced swimming test, caused LBP upregulation and steatosis, which can be rescued by NAC treatment in vivo. Taken together, our findings reveal a noncanonical mechanism of LBP as a hub for adapting to a stress-changing environment and maintaining intracellular homeostasis through physical collection of lipids and PRDX4 in-and-out LDs.

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“…The secant plane is not passing through the sarcomere' Z bands. Three different point of views of the cylinder (a, b, c); the cylinder with the cylindric section (black line) -which is an ellipse-obtained with plane slope m = 2 and z-intercept c = 1.5 (d) and the corresponding sarcomere's ultrastructural section (e).on the myofibril shape, mitochondrial structure, sarcolemmal, and sarcomere features(Burkholder & Lieber, 2001;McMillan & Eisenback, 2018;Picard, White, & Turnbull, 2013). It is generally conducted on muscular biopsy and the most informative ultrastructural images are considered those obtained from longitudinal sections of the fiber, thus oblique sections are often not considered for analysis.In this work, only through geometrical considerations, we showed that it is possible to calculate the length and the radius of the sarcomeres as well as the inclination of the secant plane starting from ultrastructural images of skeletal muscle also in the case of oblique sections.…”

mentioning

confidence: 99%

How to estimate the sarcomere size based on oblique sections of skeletal muscle

Boschi

2023

Journal of Anatomy

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Ultrastructural analysis of muscular biopsy is based on images of longitudinal sections of the fibers. Sometimes, due to experimental limitations, the resulting sections are instead oblique, and no accurate morphological information can be extracted with standard analysis methods. Thus, the biopsy is performed again, but this is too invasive and time‐consuming. In this study, we focused our attention on the sarcomere's shape and we investigated which is the structural information that can be obtained from oblique sections. A routine was written in MATLAB to allow the visualization of how a sarcomere's section appears in ultrastructural images obtained by Transmission Electron Microscopy (TEM) at different secant angles. The routine was used also to analyze the intersection between a cylinder and a plane to show how the Z‐bands and M‐line lengths vary at different secant angles. Moreover, we explored how to calculate sarcomere's radius and length as well as the secant angle from ultrastructural images, based only on geometrical considerations (Pythagorean theorem and trigonometric functions). The equations to calculate these parameters starting from ultrastructural image measurements were found. Noteworthy, to obtain the real sarcomere length in quasi‐longitudinal sections, a small correction in the standard procedure is needed and highlighted in the text. In conclusion, even non‐longitudinal sections of skeletal muscles can be used to extrapolate morphological information of sarcomeres, which are important parameters for diagnostic purposes.

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Transmission Electron Microscopy for Analysis of Mitochondria in Mouse Skeletal Muscle

Cited by 12 publications

References 16 publications

Micro‐ and nano‐tomography analysis of mouse soleus muscle using radiation

Micro‐ and nano‐tomography analysis of mouse soleus muscle using radiation

LBP resists hepatic oxidative stress by regulating LD homeostasis

How to estimate the sarcomere size based on oblique sections of skeletal muscle

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