Biological Low-Voltage Scanning Electron Microscopy
Explore the Microscopic WorldImagine being able to explore the intricate landscape of a cell's surface, navigating its mountainous organelles and valley-like contours in stunning three-dimensional detail.
For decades, scientists have dreamed of such capabilities, but biological specimens posed a particular challenge: they were too delicate to withstand the powerful beams of electrons needed to visualize them. Traditional electron microscopes often damaged the very structures researchers sought to examine, leaving them with distorted images and destroyed samples 1 5 .
High-energy electron beams cause damage to delicate biological structures, generate charging effects, and can alter the features being observed.
Low-voltage operation preserves delicate structures while providing unprecedented clarity of biological samples at the nanoscale.
At its core, scanning electron microscopy works by scanning a focused beam of electrons across a sample's surface and detecting the signals that are emitted. Traditional SEM uses high-energy electron beams, typically ranging from 5-30 kiloelectron volts (keV). While this provides detailed images, it comes with significant drawbacks for biological specimens 5 .
The high-energy electrons can penetrate deeply into the sample, causing damage to delicate structures, generating electrical charging effects that distort images, and potentially altering the very features scientists want to observe 5 .
Lower-energy electrons deposit less energy into the specimen, minimizing structural damage and preserving fine details 3 .
Less electrical charge builds up on non-conductive biological samples, decreasing distortions and artifacts 6 .
Low-energy electrons interact primarily with the sample's surface rather than penetrating deeply, providing superior surface topography 5 .
| Feature | Conventional SEM | Low-Voltage SEM |
|---|---|---|
| Operating Voltage | 5-30 keV | 1-5 keV |
| Sample Damage | Significant | Minimal |
| Charging Artifacts | Common | Reduced |
| Surface Resolution | Moderate | High (down to 1.5 nm) |
| Sample Preparation | Extensive | Simplified for some applications |
One of the most creative breakthroughs in this field came from researchers who asked a simple but profound question: Could we modify an existing SEM to function like a transmission electron microscope (TEM) while maintaining the benefits of low-voltage operation? In a 2024 study, scientists designed and tested a custom adapter that transforms a standard scanning electron microscope into a tool capable of producing TEM-like images at significantly reduced voltages 3 .
The adapter is elegantly simple in concept but sophisticated in execution. It consists of a specially designed metal plate that fits inside the SEM chamber, positioned beneath the sample holder. When the electron beam passes through an ultrathin biological sample, instead of being wasted, these transmitted electrons strike the inclined metal plate, generating secondary electrons that are then detected by the microscope's standard secondary electron detector 3 .
Researchers created a compact metal adapter only 10 mm in height (compared to the original 40 mm design) to fit physical constraints of their FEI XL-30 FEG-SEM. The plate was polished and coated with 99.99% gold foil to enhance electron interactions 3 .
Human muscle tissue samples were carefully prepared using standardized protocols: fixed in glutaraldehyde and paraformaldehyde, post-fixed with osmium tetroxide, dehydrated in ethanol, and embedded in resin before being sectioned into ultrathin slices (70 nm) using an ultramicrotome 3 .
The same sample regions were imaged using both traditional TEM (operating at 80 kV) and the modified SEM equipped with the new adapter (operating at 3-5 kV) 3 .
Researchers employed sophisticated image analysis software to quantitatively compare noise levels and texture characteristics between the two imaging methods, ensuring objective assessment of image quality 3 .
| Parameter | Traditional TEM (80 kV) | Modified SEM (3-5 kV) |
|---|---|---|
| Noise Levels | Significantly lower | Higher than TEM but acceptable |
| Sample Damage Risk | Higher due to high voltage | Reduced due to low voltage |
| Structural Clarity | Excellent | High quality, approaching TEM |
| Operational Cost | High | More cost-effective |
| Space Requirements | Significant | Compact |
"The SEM conversion device offers a practical and economical way to obtain TEM-like images from a SEM without needing a full TEM setup" 3 .
Successful biological LVSEM requires careful sample preparation to stabilize and preserve delicate structures while making them compatible with the microscope's vacuum environment and electron beam.
| Reagent/Material | Function | Application Notes |
|---|---|---|
| Glutaraldehyde | Primary fixative that crosslinks proteins | Preserves cellular structure; typically used at 2.5% concentration |
| Osmium Tetroxide | Secondary fixative that stabilizes lipids | Enhances contrast and stabilizes membrane structures |
| Ethanol or Acetone | Dehydrating agents | Gradually replaces water in samples to prepare for drying |
| Critical Point Dryer | Equipment for gentle sample drying | Prevents structural collapse during drying process |
| Gold or Platinum | Conductive coating materials | Applied to samples to prevent charging artifacts 4 |
| Silicon Chips | Mounting substrates | Provide conductive, flat surfaces for mounting samples |
| Carbon Tape | Conductive adhesive | Secures samples to mounting stubs while maintaining conductivity |
Each component addresses specific challenges in preparing biological samples for electron microscopy. The chemical fixatives stabilize delicate cellular structures in a state as close to living as possible, while the conductive coatings and proper mounting materials ensure that samples will yield clear, artifact-free images under the electron beam 4 .
A persistent challenge in biological SEM has been the distortion caused by charging effects, particularly in uncoated or frozen-hydrated samples. When the electron beam scans across non-conductive biological material, electrons accumulate, causing bright streaks, dark bands, and distorted features that obscure true biological structures. This problem is especially pronounced in lipid-rich regions like cell membranes or the myelin sheaths around nerve cells 6 7 .
In a groundbreaking 2025 study published in Nature Communications, researchers developed an ingenious solution: interleaved "leapfrog" scanning. Instead of scanning adjacent pixels sequentially (left to right, top to bottom), this novel approach skips between non-adjacent pixels in both the x and y directions, allowing time for accumulated charge to dissipate before the beam returns to nearby areas 6 .
"We went from images that were unusable due to distortion, to images where we could clearly see previously hidden structures. It has been a game-changer" - Dr. Maud Dumoux of the Rosalind Franklin Institute 7 .
Scientists are increasingly combining LVSEM with other imaging techniques, such as light microscopy or transmission electron microscopy, to gather complementary information about the same biological structures 5 .
Researchers are developing lower-cost, more compact SEM designs that could make this powerful technology available to smaller laboratories and educational institutions worldwide 9 .
Biological low-voltage scanning electron microscopy represents more than just a technical improvement in imaging technology—it offers a fundamentally gentler way of exploring the intricate architecture of life.
By reducing the energy of the electron beam, scientists can now observe biological structures with minimal damage, preserving their native state while still achieving breathtaking resolution. From customized adapters that transform existing equipment to innovative scanning patterns that eliminate distortions, the field continues to find creative solutions to longstanding challenges 3 6 7 .
As these technologies become more refined and accessible, they promise to accelerate discoveries across biology and medicine. Whether revealing the detailed organization of neural connections in the brain, capturing dynamic processes in living cells, or enabling diagnostic imaging of pathological samples, LVSEM provides a powerful lens through which we can appreciate the exquisite complexity of life at the smallest scales.