Electron microscopy requires meticulous sample preparation to visualize the ultrastructure of tissues. The process involves several crucial steps to preserve cellular components and allow for imaging at high resolution.
Key Steps in Tissue Sample Preparation for Electron Microscopy
The preparation of tissue samples for electron microscopy generally follows these steps:
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Primary Fixation: This crucial first step uses aldehydes, such as glutaraldehyde, to preserve the tissue's protein structure. Aldehydes cross-link proteins, preventing degradation and maintaining the integrity of cellular organelles.
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Secondary Fixation: Following primary fixation, osmium tetroxide is used. This step primarily fixes lipids, enhancing membrane contrast and providing better preservation of cellular membranes.
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Dehydration: Water is removed from the tissue through a graded series of solvents, typically ethanol or acetone. This is essential because water is incompatible with the resin used in the next step. The dehydration is done gradually to prevent distortion of the tissue.
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Drying/Infiltration: The dehydrated tissue is then infiltrated with a resin, such as epoxy resin. This resin embeds the tissue, providing structural support during sectioning. The resin penetrates the tissue slowly replacing the solvent. This step ensures the tissue is hardened and ready for sectioning.
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Sectioning: The resin-embedded tissue block is then sectioned using an ultramicrotome, creating extremely thin sections (typically 60-90nm) suitable for electron microscopy. These ultra-thin sections are then placed on copper grids.
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Staining (Optional): Depending on the specific application, staining techniques, such as uranyl acetate and lead citrate, can be employed to enhance contrast and highlight specific cellular components.
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Mounting: The sections on the grids are then ready to be viewed under the electron microscope.
These steps ensure the tissue is adequately preserved, allowing for clear visualization of its ultrastructure under the electron microscope. Proper preparation is critical for obtaining high-quality images and meaningful results.
