![]() 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. ![]() Boasting unmatched Z-axis resolution and accuracy, the ContourGT-K provides all of the industry recognized advantages of Bruker's proprietary white light interferometry without the deficiencies of conventional confocal and standard digital microscopes.Cellular organelle research increases understanding of the physiological functions of cells such as vital cellular processes including apoptosis, respiration, and mitosis. The gage-capable ContourGT-K provides intuitive access to an extensive library of pre-programmed filters and analyses for LED, solar cell, thick films, semiconductor, ophthalmic, medical device, MEMS and tribology applications. With exceptional roughness and 2D/3D measurement capabilities, high-resolution imaging and the industry's most advanced user friendly interface, the system offers uncompromised metrology in a simplified package with a compact footprint. Uncompromised Imaging and Metrology for Widest Range of Surfaces The ContourGT-K 3D Optical Microscope sets a new industry standard in design and cost for surface metrology performance.
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