Study Surface of Mitochondrial Membrane Using Scanning Electron Microscope (SEM)

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Wesleyan University
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Mitochondria are relatively large cell organelles scattered throughout the cytosol. They are sites of chemical reactions in which the transfer of energy to adenosine triphosphate (ATP) occurs. ATP refers to that which the majority of cells use as a mode of intracellular energy transfer also as the main energy currency. Mitochondria are known to be dynamic organelles which exhibit changes in morphology, size, localization, and mobility, which are all characteristics of their adaptive and functional changes, homeostasis and quality control. The cellular functions of mitochondria affect aging, neurodegeneration, oxidative tissue injury as well as tumorigenesis. On the other hand, these functions may also result in the damage of these organelles leading to exacerbation of disease progression particularly when excess reactive oxygen species are produced.

Among the numerous cytoplasmic components available, the particular interest in the study of mitochondria has gained increased attention not only because of their role in transferring energy from organic compounds to ATP, but also the distinctive visibility of its components. The visibility with which the living components of mitochondria can be observed is made more vivid using either the vital staining procedures or contrast-increasing devices. With adequate fixation and staining, the contents of mitochondria are readily observable hence their general consideration in microscopy studies. The biochemical studies involving this organelle have been aided by the development of separatory techniques which have presented valuable materials for these studies. Unless one assumes how the components of mitochondria are organized, it is difficult to explain the biochemical findings of these biochemical processes satisfactorily. For instance, some biochemists have found out that there is a definite spatial relationship among the organelles, making their behaviors to resemble highly integrated enzymatic systems. It is only under a light microscope that the organization and relationship of the components are not clear as mitochondria appear as homogenous and structure-less bodies. That is so because of the limited resolution power of the light microscope (Tanaka & Mitsushima, 1984).

Electron Microscopy and Scanning Electron Microscope (SEM)

When viewed under the electron microscope, the organization, structure and chemical reactions can be observed when the specimen is thin enough for an examination. Electron microscopes use accelerated beams of electrons as a source of illumination making it have higher resolution power than the light microscope. Since its inception, electron microscopy has been a vital tool in medical sciences. The advances in the field have been inspired by the need to find the best methods of preservation and analysis of structures while at the same time keeping them close to their natural or native state. In as much as little to no attention has been given to wet samples under the view of being impractical, fully hydrated sample can be observed at body or room temperatures. Thus, many artifacts of the preparation of samples can be eliminated to grant routine and reproducible imaging.

Scanning electron microscope (SEM) is a variety of electron microscope which creates an image sample by scanning the surface of a mitochondrion with focused electron beams (Palade, 1953). When focused on the sample, the atoms from the surface of the specimen get to interact with the electrons thereby providing information on the nature, composition, and topography of the mitochondrial surface. A pattern known as a raster scan is used to focus the electron beam whose signal information is combined with its position for an image to be produced.

Mitochondrial Structure

The compartment containing the mitochondrial DNA is enclosed by double membranes when observed with detail by scanning electron microscope (Sumire, Hitoshi & Keiichi, 1991). The morphological variation of the cristae is so because of how the topology of the inner membrane is dynamically controlled. The outer membrane, on the other hand, limits the operations of mitochondria by acting as the boundary. A mitochondrions size is another morphological aspect that usually changes through the processes of both fission and fusion. Important to note with the two processes is that any anomaly in the fission process results in the formation of giant mitochondria while abnormality in the fusion process causes the formation of fragmented ones.

In some circumstances under certain chemicals, mitochondria can assume spherical shapes. For instance, when used, Carbonyl cyanide m-chlorophenylhydrazone (CCCP) is often responsible for depolarization and fragmentation thereby affecting the functions of mitochondria as well as triggering various rapid responses (Picard, White & Turnbull, 2012). On the other side, CCCP can cause unique mitochondrial structural formation resulting in their spherical shapes. This shape makes it possible for membranes to enwrap cytoplasm as well as other organelles within the mitochondrion. An indication of various tomographic studies and Electron serial sectioning asserts that the structures are almost completely delimited and that the lumen is connected to the cytosol only by a small orifice that is present. This structuring represents just one of the morphological dynamics of mitochondria when observed under scanning electron microscope.

In relationship to the use of CCP to analyze the spherical shapes of mitochondria, Ding et al. (2012) found out that a large number of mitochondria they studied had c-shaped or ring-shaped morphology. To concur with the outcomes of other studies, they discovered that the mitochondria enclosed various cytosolic components and that they had a ball-like shape. The lumen contained cytosolic materials and was surrounded by double membranes. Worth noting is the fact that the external cytoplasm connects to the lumen via a small opening. The membranes topology, from electron tomographic reconstruction, demonstrated vesicular configuration of mitochondrial structures. Basically, there were the inner and the outer membranes. After treatment with CCP, mitochondrial spheroids formed as a response to oxidative mitochondrial damage. The damage was in no way connected to mitophagy, that is, the selective process in which stressed defective mitochondria degrade by themselves.

Technological advancement is credited with improving how samples are being viewed under electron microscopes. Notably, the recent adaptation of scanning electron microscope in the observation of hydrated samples has improved how differential pumping capabilities, as well as that of detectors, support the survival of these samples in a vapor environment. The main reason for imaging wet fully-fluid cell is such that the image resolution is sharp and contrasting when the cell is open. Once this has happened, the components can easily be studied.

Mitochondrial Morphology, Topology, and Functionality

Various scientists (Thiberge et al., 2004) have conducted numerous experiments to test some aspects such as functionality, morphology, and topology of mitochondria. These experiments have involved particular materials and methods which are all meant to test specific aspects. The results, as will be seen, will be consistent irrespective of the area of the body from which the mitochondria is extracted. The physiological conditions of wet samples can be maintained with imaging resolution better than the resolution of standard Scanning electron microscope (SEM). This kind of scanning is dependent on the thinness of the membranous samples. The membrane protecting the sample components has to be thin to be transparent when interacting with the beam of electrons. Initially, that is, before the idea of the wet SEM imaging system came by, some cellular observations could not be observed under the standard SEM. When it was discovered, this imaging system has enabled how organelles like mitochondria and their internal structures are viewed. Additionally, gold immunolabeling and staining can be imaged even without drying and coating. Through SEM, not only the structural connectivity of tissues but also the extracellular activities can be viewed. This can be done via various methods including chamber enclosure, staining methods, resolution, and CL imaging.

Methods and Process

The Chambers method involves placing the mitochondrion sample in an enclosed window consisting of a thin electron-transparent membrane that acts as a partition. Beams of electrons are then directed towards the partition membrane to interact with the sample before scattering above on a backscattered electron (BSE) detector. Of the membranes that have been tested through this procedure, it has been found that the most appropriate is the polyimide membranes of 145 nm in thickness. The insertion techniques have been improved so much that adherent cultured cells can now be grown directly on any partitioned medium used. When grown on suspension, any cell whether mammalian, bacteria or any other type can be adsorbed or centrifuged on the particular membrane before imaging. After that, mechanical or manual procedures can be used to bring tissues to direct contact with the membrane. Another improvement involves introducing a leakage hole into the electron microscope chamber; calculations done on this hole are enough to ensure it keeps the sample at low temperatures while keeping it fully hydrated at the same time. This process lowers membranous strain and enhances its endurance. Also, it increases resolution as ultrathin membranes of as low as 50 nm can be used. The affinity of the cells can be improved by the treatment of polyimide membrane with fibronectin (Thiberge et al., 2004).

The Staining technique involves fixing cells and tissues with chemicals such as paraformaldehyde and glutaraldehyde then rinsing them in cacodylate buffer containing sucrose. When staining is done using uranyl acetate, the samples have to be rinsed in water containing tanning acid. Osmium tetroxide is also used as a staining agent then washing the samples in water so as to view them in a wet state.

When it comes to resolution, there is complete dependence on the BSE volume, energy or acceleration voltage of the beams of electrons. The larger the volume of the scattered electrons the larger the signals produced. The high contrasts resulting to higher resolution is determined by the difference between the electrons that scatter back after just a little interaction and those that probe the sample further. When the mitochondrion sample is unfixed, the resolution is still possible from above 20 nm because the intercellular viscosity lowers the cells thermal motion. It has also been found that emulsions produce strong signals because they have more materials towards the surface and deform when wetting the mitochondrial membranes.

Apart from fixation, other advances in the study of the surface of mitochondria include embedding and sectioning. Literature from other cytologists indicates that the study of mitochondria brings almost similar results irrespective of the source of the organelles. Mammalian materials or specimen are commonly tested for mitochondrial structure. These animals include rats, mice, and rabbits. Before examination via the electron microscope, the samples are fixed, embedded and then sectioned. It is worth noting that, for the structural clarity to be observed, the thinness of the section used has to be between 0.1 and 0.05u.

Cristae mitochondriales are particularly the structural features that make possible the identification of mitochondria in electron microscopy when staining fai...

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