Low-voltage electron microscope (LVEM) is an electron microscope which operates at accelerating voltages of a few kiloelectronvolts or less. While the low voltage electron microscopy technique will never replace conventional high voltage electron microscopes, it is quickly becoming appreciated for many different disciplines. There are some significant advantages to imaging under LVEM that allow high quality images to be produced for samples that would be otherwise impossible to visualize under conventional electron microscopy techniques.
Currently there is only one commercially available low voltage transmission electron microscope.[1] While its architecture is very similar to a conventional transmission electron microscope, it has a few key changes that enable it to take advantage of a 5 keV electron source. Its electron column is inversely mounted, namely the source is at the bottom of the instrument. In TEM mode, the electrons are directed up through the sample and form a pinpoint image on a mono-crystalline YAG screen. Light objectives are then used to magnify the image further to the CCD camera. The column has internal detectors for measuring backscattered electrons for imaging in the SEM mode. The instrument also includes a photomultiplier used to image in the STEM mode.
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A substantial decrease of electron energy allows for a significant improvement of contrast of light elements. The comparison images below show that decreasing the acceleration voltage from 80 kV to 5 kV significantly enhances the contrast of test samples. The improved contrast is a direct result of increased electron scattering associated with a reduced accelerating voltage.
LVEM brings an enhancement of imaging contrast nearly twenty times higher than for 100 kV. This is very promising for biological specimens which are composed from light elements and don't exhibit sufficient contrast in classical TEMs.[2]
Further, a relatively low mean free path (15 nm) for organic samples at 5 kV means that for samples with constant thickness, high contrast will be obtained from small variations in density. For example, for 5% contrast in the LVEM bright field image, we will only need to have a difference in density between the phases of 0.07 g/cm3. This means that the usual need to stain polymers for enhanced contrast in the TEM (typically done with osmium or ruthenium tetraoxide) may not be necessary with the low voltage electron microscopy technique.[3]
| Comparison – TEM images of unstained thin section of rat heart | |||||||||
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The improved contrast allows for the significant reduction, or elimination, of the heavy metal negative staining step for TEM imaging of light elements (H, C, N, O, S, P). While staining is beneficial for experiments aimed at high resolution structure determination, it is highly undesirable in certain protein sample preparations, because it could destabilize the protein sample due to its acid pH and relatively high heavy metal concentration. The addition of stain to sectioned samples such as biological materials or polymers can also introduce imaging artifacts.
LVEM experiments carried out on an extracted membrane protein sample that was analyzed with and without the staining procedure show a marked improvement in the appearance of the sample when standard staining is omitted. Results show that LVEM could be even more useful than conventional EM for this particular application because it avoids the potentially disrupting staining step, thus providing an undisturbed image of the protein’s aggregation state.[4][5]
Additionally, The ability to eliminate the staining step could aid to improve safety in the lab, as common heavy metal stains, such as uranyl acetate do have associated health risks.
Both transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are possible on the same instrument. It is even possible to have the scanning transmission electron microscopy (STEM) as well. This is not possible on conventional electron microscopes since SEM and TEM are normally carried out at very different accelerating voltages.
Present low voltage electron microscopes are capable of spatial resolutions of about 2.5 nm in TEM 2.0 nm in STEM and 3.0 nm in SEM [3]
Reduced accelerating voltages allows a significant reduction in the column size required for the microscope. This allows it to be packaged into a convenient benchtop format.
A miniaturized electron column is inherently less sensitive to external vibration and noise. This could be a significant advantage over conventional electron microscopes since specially isolated facilities are not required for its installation.
Currently available low voltage microscopes are only able to obtain resolutions of 2–3 nanometers. While this is well beyond resolutions possible from optical (light) microscopes, they are not yet able to compete with the atomic resolution obtainable from conventional (higher voltage) electron microscopes.
Low voltage limits the maximum thickness of samples which can be studied in the TEM or STEM mode. Whereas it is about 100–200 nm in conventional TEM, it decreases to around 20–65 nanometers for LVEM. However, thicknesses of the order of 20 nm or less are required to attain the maximal resolution in the TEM and STEM modes.[2][3] These thickness are achievable with the use of an ultramicrotome.
LVEM is especially efficient for the following applications.