Overview
Electron microscopy (EM) was developed to overcome the resolution limits of light microscopy, which cannot resolve structures smaller than about 0.2 micrometres. By using a focused electron beam instead of light, transmission electron microscopes can achieve resolutions as fine as 0.05 nanometres.
This lesson covers the working principles of the transmission electron microscope (TEM) and scanning electron microscope (SEM), the specialised tissue processing steps required for EM (fixation, dehydration, embedding, ultrathin sectioning), and the clinical applications of EM in modern histopathology.
Learning Objectives
After this lesson you will be able to…- Explain the working principle of the transmission electron microscope (TEM)
- Differentiate the transmission and scanning electron microscopes
- Describe the tissue processing steps required for electron microscopy
- Explain the role of double fixation using glutaraldehyde and osmium tetroxide
- List the clinical applications of electron microscopy in diagnostic pathology
Clinical Story
Why This MattersA child presents with nephrotic syndrome and a renal biopsy shows minimal changes on light microscopy. To confirm the diagnosis and exclude other glomerular diseases, a portion of the biopsy is processed for electron microscopy, where diffuse podocyte foot process effacement — visible only at the ultrastructural level — confirms minimal change disease.
Core Concepts
The TEM is the direct counterpart of the light microscope but uses a high-voltage electron beam instead of light, accelerated by an anode and focused by electromagnetic lenses. The beam passes through an ultrathin specimen, and the resulting image—formed by differential absorption and scattering of electrons—is projected onto a fluorescent screen or recorded photographically.
The SEM produces images of surface topography rather than internal structure. An electron beam scans across the specimen surface, and secondary and backscattered electrons ejected from the sample are collected by detectors and converted into a television-like image, making it ideal for studying unsectioned, solid specimens.
EM tissue processing involves fixation, dehydration, embedding, ultrathin sectioning, and positive staining with heavy metals. Double fixation—first in glutaraldehyde, then in osmium tetroxide—is the most popular method, since glutaraldehyde stabilises proteins while osmium tetroxide additionally fixes lipids that would otherwise be lost during dehydration.
Laboratory Principle
Electron microscopy exploits the extremely short wavelength of accelerated electrons compared to visible light, allowing far greater resolving power. As the electron beam passes through (TEM) or is scattered from the surface of (SEM) an ultrathin or metal-coated specimen, the pattern of absorption, scattering, or secondary electron emission encodes structural information that is converted into a magnified image, revealing sub-cellular detail invisible to light microscopy.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Glutaraldehyde | 2.5% buffered solution | Primary fixation for EM, stabilises proteins | 4°C, prepare fresh |
| Osmium tetroxide | 1-2% buffered solution | Secondary (post) fixation, preserves lipids | 4°C, handle with caution (toxic/volatile) |
| Epoxy resin | With curing agent as per manufacturer | Embedding medium for ultrathin sectioning | Room temperature until cured |
Step-by-Step Procedure
Cut 1-2 mm thick tissue slices immediately after interruption of blood supply and transfer to glutaraldehyde fixative.
Following primary fixation, post-fix in osmium tetroxide to stabilise lipids that glutaraldehyde alone cannot preserve.
Dehydrate the tissue through an ascending ethanol series (40% to 100%) to remove all free water.
Transfer the dehydrated tissue (via a transitional fluid such as epoxypropane if needed) into bubble-free epoxy resin within polythene capsules or moulds and cure.
Cut ultrathin sections (approximately 60 nm) on an ultramicrotome using glass or diamond knives, then positively stain with heavy metals before examination.
Flow Diagram
Quality Control
Confirm adequate fixation timing (glutaraldehyde penetrates at 2-3 mm/hour, osmium tetroxide at 1 mm/hour) and check section thickness (~60 nm) under the electron microscope before full diagnostic examination.
Where possible, participate in EM proficiency schemes for renal or muscle biopsy interpretation, since ultrastructural diagnosis (e.g. glomerular basement membrane changes) often requires specialised external validation.
Reference Values
Normal Ranges⚠️ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Diffuse podocyte foot process effacement on EM | Consistent with minimal change disease or early focal segmental glomerulosclerosis | Correlate with light microscopy and immunofluorescence findings |
| Electron-dense deposits along glomerular basement membrane | Suggests immune complex-mediated glomerulonephritis | Correlate with clinical and serological findings; further sub-classification as indicated |
| Normal ultrastructure despite proteinuria | May indicate a functional or non-structural cause | Consider alternative diagnoses and further clinical workup |
Common Errors & How to Avoid Them
Cause: Delayed fixation after tissue removal, allowing autolysis to begin before glutaraldehyde penetrates.
Prevention: Fix tissue immediately after excision, cutting it into thin (1-2 mm) slices for rapid fixative penetration.
Cause: Glutaraldehyde alone used without osmium tetroxide post-fixation.
Prevention: Always perform double fixation with osmium tetroxide following glutaraldehyde for optimal membrane preservation.
Cause: Ultramicrotome or knife not properly calibrated, producing sections thicker than the required ~60 nm.
Prevention: Use a properly maintained ultramicrotome with sharp glass or diamond knives and verify section thickness before full examination.
Laboratory Tips from the Bench
Always cut EM tissue into very thin slices (1-2 mm) immediately after removal — glutaraldehyde penetrates slowly, and delay risks autolytic change.
Never skip osmium tetroxide post-fixation when lipid membrane preservation matters, such as in renal or nerve biopsies.
Remember: 'Glutaraldehyde for protein, Osmium for fat' — the two-step double-fixation strategy that makes EM tissue preservation possible.
Important Notes
Osmium tetroxide is volatile and toxic, requiring careful handling with appropriate ventilation and personal protective equipment.
Despite advances in immunohistochemistry and molecular pathology, electron microscopy remains indispensable for diagnosing certain glomerular diseases, ciliary dyskinesia, and some inborn errors of metabolism.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your KnowledgePresents with generalised oedema and heavy proteinuria. Renal biopsy shows unremarkable glomeruli on light microscopy; a portion is processed for electron microscopy.
The combination of normal light microscopy, negative immunofluorescence, and diffuse foot process effacement without electron-dense deposits on electron microscopy is diagnostic of minimal change disease, the most common cause of nephrotic syndrome in children.
- →Electron microscopy is essential when light microscopy and immunofluorescence are unremarkable in nephrotic syndrome.
- →Foot process effacement without deposits is the hallmark of minimal change disease.
- →EM findings guide both diagnosis and treatment (steroid responsiveness) in paediatric nephrotic syndrome.
Frequently Asked Questions
Glutaraldehyde stabilises proteins effectively but does not adequately fix lipids, which can be extracted during subsequent dehydration steps — hence the need for osmium tetroxide post-fixation.
TEM produces a two-dimensional image of internal ultrastructure by transmitting electrons through a thin section, while SEM produces a three-dimensional-appearing image of surface topography by detecting scattered electrons from a solid specimen.
Glutaraldehyde penetrates tissue slowly (about 2-3 mm per hour), so thin slices ensure the fixative reaches the centre of the tissue before autolytic changes can occur.
Quick Revision
10-Minute ReviewKey Takeaways
- Electron microscopy overcomes the resolution limits of light microscopy using electron beams.
- TEM and SEM serve different purposes — internal ultrastructure versus surface topography.
- Double fixation with glutaraldehyde and osmium tetroxide preserves both proteins and lipids.
- Ultrathin sectioning at ~60 nm requires a specialised ultramicrotome.
- EM applications include kidney and skin biopsy interpretation, viral infections, and tumour diagnosis.
- Prompt, thin-slice fixation is critical to avoid autolytic ultrastructural artefact.
Competency Checklist
Track Your MasteryReferences
- NIOS Histology and Cytology Practical Manual, Lesson 19: Electron Microscopy.
- Bancroft JD, Gamble M. Theory and Practice of Histological Techniques. 7th ed.
- Dalton AJ, Haguenau F. Ultrastructure of the Kidney. Academic Press.