Introduction

Bioimaging, the visualization of biological structures and processes, has revolutionized various fields such as medicine, biology, and pharmacology. However, behind the stunning images lies a complex process of sample preparation, including embedding and sectioning, which is crucial for obtaining accurate and high-quality results. In this article, we'll delve into the fundamentals of sample processing for bioimaging in a way that's accessible to all.

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Understanding Sample Processing

Figure 1. Schematic diagram illustrating the procedure for embedding large organs. (Zhanmu O, et al.; 2020)

Sample processing involves a series of steps aimed at preparing biological samples for imaging under a microscope. This process is vital as it helps preserve the structure and integrity of the sample while enhancing contrast and resolution for imaging. Here's a breakdown of the key components:

• Sample Fixation

The process begins with sample fixation, where biological specimens are treated with fixatives to preserve their structure and prevent degradation. Common fixatives include formaldehyde, glutaraldehyde, and paraformaldehyde, which cross-link proteins and other biomolecules, stabilizing the cellular architecture.

• Dehydration

Following fixation, samples are dehydrated to remove water and replace it with a solvent that can penetrate the tissue without disrupting its structure. Ethanol and acetone are commonly used dehydrating agents, gradually replacing water molecules in the sample.

• Clearing

After dehydration, samples may undergo a clearing process to render them transparent, allowing light to pass through easily during imaging. Clearing agents such as xylene or benzyl alcohol/benzyl benzoate are employed to remove residual fixatives and further enhance transparency.

• Embedding

Embedding is a critical step where dehydrated and cleared samples are embedded in a solid medium to provide support and facilitate sectioning. This is typically achieved using paraffin wax or resin-based embedding compounds, ensuring the sample is securely held in place for precise sectioning.

• Sectioning

Once embedded, samples are sliced into thin sections using specialized equipment such as microtomes or cryostats. Microtomy involves cutting sections of fixed samples embedded in paraffin wax, while cryosectioning is suitable for frozen specimens embedded in cryoembedding media. The thickness of sections can vary depending on the specific requirements of the imaging technique and the sample type.

• Mounting

Following sectioning, samples are mounted onto suitable substrates such as glass slides or coverslips for imaging. Mounting media, such as mounting medium with DAPI (4',6-diamidino-2-phenylindole) for nuclear staining or fluorescent mounting medium for fluorescence microscopy, are applied to enhance contrast and preserve the samples during imaging.

• Staining

Staining is often employed to enhance contrast and visualize specific structures or molecules within the sample. Various staining techniques, including hematoxylin and eosin (H&E) staining for general histology or immunofluorescence staining for specific protein detection, are used depending on the imaging modality and the information required.

• Quality Control

Throughout the sample processing workflow, quality control measures are essential to ensure the integrity and reliability of the imaging data. This includes assessing the quality of fixation, section thickness, staining intensity, and overall sample morphology to identify any potential issues that may impact imaging results.

Conclusion

Sample processing is a critical aspect of bioimaging that requires careful attention to detail and precision. By following established protocols and techniques for sample fixation, embedding, sectioning, and staining, researchers can prepare high-quality samples for imaging studies, providing valuable insights into biological structures and processes. Understanding the fundamentals of sample processing not only enhances the quality of imaging data but also contributes to advancements in various fields, from basic research to clinical diagnostics and beyond.

1. Zhanmu O, et al.; Paraffin-embedding for large volume bio-tissue. Sci Rep. 2020, 10(1):12639.

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