Samples are prepared for light microscopy through a series of steps that typically involve fixing, embedding, sectioning, and staining to preserve cellular structures and enhance visibility.
The specific steps involved can vary depending on the type of sample and the desired outcome, but the general process includes the following:
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Fixation: This process preserves the tissue or cells, preventing degradation and maintaining their structure. Common fixatives include formaldehyde (formalin) and glutaraldehyde. Fixation can be achieved through chemical means (immersion or perfusion) or physical methods (freezing).
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Dehydration: Water is removed from the sample, typically through a series of increasing concentrations of ethanol. This prepares the tissue for embedding in a non-aqueous medium.
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Clearing: Ethanol is replaced with a solvent that is miscible with both ethanol and the embedding medium. This step makes the tissue transparent. Common clearing agents include xylene and toluene.
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Embedding: The sample is infiltrated and surrounded by a solid medium, such as paraffin wax or resin, to provide support during sectioning. This creates a block that can be thinly sliced. Embedding facilitates sectioning.
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Sectioning: The embedded sample is sliced into thin sections using a microtome. The thickness of the sections depends on the sample and the research question but is typically 2-5 micrometers for routine histology. The specimen is cut into thin sections.
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Mounting: The thin sections are placed onto glass slides for viewing. An adhesive may be used to ensure the section adheres to the slide.
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Staining: Stains are applied to the sections to enhance contrast and highlight specific cellular components. Common stains include hematoxylin and eosin (H&E), which stain different cellular structures different colors. Other stains are specific to certain molecules or cell types.
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Coverslipping: A thin glass coverslip is placed over the stained section using a mounting medium. This protects the sample and improves image quality.
In summary, sample preparation for light microscopy is a crucial process that ensures the preservation, visibility, and accurate analysis of biological specimens. These steps allow researchers and medical professionals to examine cellular structures and identify abnormalities.
