Low Voltage Electron Microscopy for Negatively Stained Materials

Low Voltage Electron Microscopy (LVEM) enhances contrast, streamlines imaging, and simplifies staining procedures for negatively stained materials.

In Transmission Electron Microscopy (TEM), contrast formation depends on the differential transmission and scattering of the electron beam by the sample, which is determined by the density, or atomic composition, of the sample. Biological samples are often composed of low atomic weight elements, resulting in weak scattering and poor contrast at high accelerating voltages. To compensate, electron microscopists traditionally rely on negative staining to artificially enhance contrast.

Negative staining is a widely used technique in transmission electron microscopy for imaging biological low-density particulate materials such as liposomes, proteins, and viruses, as well as other soft non-biologics like polymeric nanoparticles. This technique works by enveloping specimens in an electron-dense stain, typically heavy metal salts like uranyl acetate (UA), to enhance contrast and reveal structural detail. Importantly, the resolution in negatively stained samples is usually limited not by the microscope, but by the grain size of the stain itself (Gallagher et al., 2019.)

LVEM is well-suited for imaging of these soft materials using classic negative stain techniques, providing high quality image output. For example, negatively stained small viruses, such as flaviviruses, have been successfully imaged with LVEM, with fine ultrastructural details well below 10 nm clearly visible (Möller et al., 2020). A major advantage of LVEM is that micrographs often require little or no additional contrast enhancement to visualize critical structural features.

The Delong Instruments LVEM 5 and LVEM 25E offer several additional advantages to samples typically requiring negative stain workflows such as, higher intrinsic contrast, smaller lab footprint, reduced consumable costs, and simpler sample preparation requirements. As a result of the lower accelerating voltage, LVEM uniquely provides flexibility when imaging these materials, enabling high-contrast imaging with low density and safer alternative stains or by even eliminating heavy metal staining altogether for certain applications. This greatly simplifies sample preparation, minimizes occupational hazards, and reduces potential image artifacts. These features make LVEM especially useful in research environments where imaging quality, ease of use, and safety, are priorities.

In summary, LVEM provides a practical, cost-effective, and efficient alternative to high-voltage TEM for imaging of materials typically requiring a negative-stain preparation. The LVEM 5 and LVEM 25E offer many advantages, such as improved image contrast, lessened occupational hazards and significantly reduced capital and operational costs, along with a much smaller lab footprint. Expanding the use of LVEM in research and diagnostic applications can improve accessibility to high-quality electron microscopy without the constraints associated with high-voltage systems.

Gallagher, J. R., Kim, A. J., Gulati, N. M., & Harris, A. K. (2019). Negative-stain transmission electron microscopy of molecular complexes for image analysis by 2D class averaging. Current Protocols in Microbiology, 54(1), e90. https://doi.org/10.1002/cpmc.90

Möller, L., Holland, G., & Laue, M. (2020). Diagnostic electron microscopy of viruses with low-voltage electron microscopes. Ultrastructural Pathology, 44(6), 535–544. https://doi.org/10.1369/0022155420929438