Transmission electron microscopy (TEM) enables the visualization of neural circuits and synaptic connections at nanometer-scale resolution. Dr. Concepcion Lillo Delgado leverages TEM, alongside immunocytochemistry, to precisely identify cellular structures and molecular markers, advancing insights into diseases and cellular mechanisms in neuroscience.

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1.   Why use this method?

Transmission electron microscopy allows the visualization of cells, subcellular structures, and proteins in biological samples at a nanometer-scale resolution. This technique reveals levels of detail and complexity that are inaccessible by optical microscopy due to the utilization of a focused beam of high-energy electrons. The detection of electrons transmitted through the sample leads to the formation of a high-resolution image. In neuroscience, transmission electron microscopy enables the study of synaptic connections, detailed mapping of neural circuits, and identification of protein-level changes in models of neurodegenerative diseases. Additionally, in tandem with immunocytochemistry, this method allows precise examination of targeted organelles or molecular markers within the cell, validating observations made using optical techniques.

2.  What you’ll need

Equipment

• Transmission Electron Microscope:
  • Electron gun and high voltage source
  • High-vacuum system – maintains the vacuum levels throughout the microscope column required to produce a consistent electron beam
  • Microscope column – consists of lenses focusing electron beam onto the sample
  • Detectors – capture images in a digital format
  • Control computers and software
• Ultramicrotome – enables ultra-thin sample sectioning

Reagents:

• Fixatives: glutaraldehyde (2.5%) and paraformaldehyde (4%), osmium tetroxide (1%)
• Dehydration agents: series of ethanol solutions (70°, 90°, 96°, 100°), propylene oxide
• Embedding media: epoxy resin: propylene oxide 1:1
• Counterstaining: uranyl acetate and lead nitrate
• Immunocytochemistry:
  • electron dense substances (e.g. colloidal gold, diaminobenzidine)
  • primary antibodies specific to the targeted protein
  • gold-conjugated secondary antibodies

Other:

• Imagining software
• Fume hood for handling toxic chemicals
• Diamond or glass knives for ultramicrotome

3. Step-by-step instructions

 1.Sample preparation

• Conventional: Fix the tissue in the glutaraldehyde and paraformaldehyde at 4°C for 2-4h. Then wash and treat it with osmium tetroxide for 1h. Gradually dehydrate the tissue using graded series of ethanol solutions, then transition sample to propylene oxide solution. Infiltrate the tissue with epoxy resin and polymerize at 60°C for 48h. Afterwards, cut ultra-thin sections (60-90 nm) of the tissue using ultramicrotome. Counterstain the samples using uranyl acetate and lead nitrate.
• With immunolabeling:
  • Pre-embedding: Mildly fix the tissue using glutaraldehyde and paraformaldehyde. Incubate the tissue in blocking solution, then treat it with primary antibodies and gold-conjugated secondary antibodies, respectively. Stabilize the antibodies by fixing the sample with glutaraldehyde. Then, continue the process as described in the conventional sample preparation.
  • Post-embedding: Fix the sample using chemical fixation or cryofixation technique. Mildly dehydrate the tissue and embed it in hydrophilic resin using UV polymerization. Section the tissue into ultra-thin slices using ultramicrotome and load them onto grids. Incubate the samples in a blocking solution, then treat them with primary antibodies and gold-conjugated secondary antibodies, respectively.

2.Imaging

• Load prepared grid into the specimen holder and then into the transmission electron microscope.
• Adjust imagining parameters for optimal resolution and contrast.
• Capture high-quality images of the tissue or labeled structures/ proteins.

4. Practical tips

• Handle samples carefully to avoid damaging tissue during processing and introducing artifacts.
• Avoid combining counterstaining with immunolabeling due to the high probability of obscuring the marked proteins.
• Always ensure that the microtome knives are sufficiently sharp to produce smooth, even slices.

5.  Critical appraisal & implications for future research

Transmission electron microscopy coupled with immunolabeling is a powerful tool for identifying neuronal structures, such as synapses, dendritic spines, and axons, expanding our understanding of neurophysiology at the nanometer-scale. However, expensive equipment and maintenance, along with challenging sample preparation and requirement for skilled personnel, limit the use of electron microscopy. Subsequent research should prioritize the simplification of slice preparation and general operation of the electron microscope. While electron microscopy provides a high resolution, it can capture a relatively small area in a single image. Therefore, it is vital to integrate this technique with novel computational tools to improve the imaging, reconstruction, and analysis of large-scale neural networks.

This protocol is licensed under a Creative Commons Attribution-NonCommercial (CC BY-NC) license, allowing sharing and adaptation for non-commercial purposes with proper attribution.