![]() 3D reconstruction is especially important for measurements associated with the folds of the inner mitochondrial membrane of mitochondria known as cristae. This is especially true for ultrastructure such as nanotunnels. However, such methods are ultimately only estimations while 3D reconstruction offers a more accurate representation of the true subcellular structures. Point counting is traditional used for these 2D micrographs, and is a powerful method for obtaining volume. TEM typically has resolution of less than 50 pm whereas the resolution for SEM images are 0.5 nm additionally, TEM allows for magnifications nearly 20x higher than SEM. In many cases, 2D TEM can be more advantageous than SEM. Įmerging imaging technologies have facilitated visualization of organelles and tissues resulting in high-resolution 3D reconstruction. As opposed to some other 3D methods of reconstruction, SBF-SEM can generate thousands of individual images allowing for higher resolution and detection of specific changes in organelle morphology. In SBF-SEM, sectioning is automated and typically involves heavily mordanted samples allowing for backscatter detection and large depths of sectioning of the block face in the z-plane. and has become an established technique to gather volumes of data from various biological samples. SBF-SEM is a relatively new technique that was developed at the Max Planck Institute in 2004 by Horstmann et al. This protocol utilizes SBF-SEM for several reasons. While these are the most common methods, other EM imaging techniques such as automated tape-collecting ultramicrotome scanning electron microscopy (ATUM-SEM) may also be employed. FIB-SEM and SBF-SEM are two commonly used methods for generating EM data for 3D reconstruction and both are viable options for the protocol described here. The high-resolution stack of images that is generated can in turn provide for unprecedented visualization and analysis of ultrastructure in 3D. With an in-chamber ultramicrotome (SBF-SEM) or ion beam (FIB-SEM), samples can thus be continuously sectioned and the block face imaged through very large volumes in the z-plane. The novel benefit of volume electron microscopy is the ability to generate near-TEM resolution images using backscatter detection of a block face rather than from an ultrathin section. TEM, alternatively, produces nanometer-resolution images by transmitting electrons through an ultrathin section of a sample. SEM utilizes high-resolution back-scatter detection to provide detailed information of the surface of a sample. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are the most common types of electron microscopy used to study organelle ultrastructure. Despite advances in light microscopy imaging in recent years, EM continues to provide unmatched high resolution ultrastructural detail and 3D visualization. ![]() Imaging of mitochondria and ER has typically been performed using light microscopy and electron microscopy (EM). Because mitochondria and ER are crucial for cellular function and survival, their study is relevant to many disciplines and they have potential as targets for pharmaceutical research on neurodegenerative, cancer, and viral disease treatments. Given the role of mitochondria in apoptosis, which is a crucial process involved in cancer, mitochondrial research is critical for cancer treatment. Further, calcium levels play a role in regulation of the citric acid cycle and calcium signaling associated with apoptosis. In addition, the mitochondrial role in maintenance of calcium homeostasis is connected to cellular apoptosis cellular calcium levels affect ER calcium levels, which in turn regulate mitosis. For example, Dynamin-related protein-1 (DRP-1) regulates mitochondrial fission and associates with early stages of apoptosis, and changes in mitochondrial ultra-structures are associated with chemical pathways that regulate calcium, potassium, and other biomolecules. Mitochondria are typically associated with their role in oxidative phosphorylation, which is crucial for ATP generation, however, their functions extend beyond energetics. ![]() Because these organelles play major roles in regulating homeostasis and ensuring organism survival, it is important to study their various structure-dependent functions. ![]() Organelles involved in metabolism, such as mitochondria and endoplasmic reticulum (ER), are some of the most studied cellular structures. Cellular organelle research increases understanding of the physiological functions of cells such as vital cellular processes including apoptosis, respiration, and mitosis. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |