Low Voltage Electron Microscopy Allows Flexibility in the Staining of Biological Thin Sections

Low Voltage Electron Microscopy (LVEM) offers an improved method for obtaining high-contrast micrographs of biological thin sections, providing greater flexibility in sample preparation. Operating at a nominal accelerating voltage of 25kV, the LVEM 25E leverages the fundamental principles of z-contrast, enabling clear imaging of even unstained specimens - a feat not possible in conventional instruments. Through comparative analysis with three staining protocols, LVEM demonstrates its ability to generate superior quality micrographs while mitigating the drawbacks of conventional staining techniques.

When pathological specimens are placed under the beam of an electron microscope (EM), ultrastructural abnormalities existing at the nanometric scale appear. The magnification and resolution afforded by electron microscopy is therefore invaluable.

In renal samples for example, only a few hundred nanometers would distinguish a glomerulus’ normal basement membrane from an abnormally thin one. In the evaluation of lung’s cilia, the observation of microtubules or dynein arms only several nanometers in length might serve as key diagnostic markers.

While the micro- and nano-scaled information provided by electron microscopy is often essential to clinical diagnoses, the steps required to prepare biological tissue thin sections for EM observation are numerous and at times laborious.

From the moment tissue is obtained from a biopsy, multiple steps are required to preserve its integrity. Although subject to variation, this process begins by fixing the sample in glutaraldehyde and post-fixing in osmium tetroxide.

The sample is then dehydrated with successive baths of graded ethanol and propylene oxide, until the sample can be embedded in resin. Once an embedded block of tissue is obtained, thick sections can be prepared in order to grossly locate the tissue of interest.

These thick sections are then further sectioned down to 50-200nm for optimal electron beam transparency before they can be adhered to TEM grids. Finally, the sample is stained with heavy metal solutions that will ensure sufficient contrast for imaging.

In transmission and scanning transmission electron microscopy (TEM/STEM), the microscope’s electron beam passes through the sample and its electrons are scattered by the specimen to varying degrees.

In some areas of the specimen, the beam electrons will pass through unaffected and will be collected by the instrument’s detector. These areas will be represented in the image with high brightness. Reciprocally, other areas of the specimen will completely scatter the beam electrons, preventing them from reaching the detector. These areas are then represented as dark regions in the image. Finally, in most areas, some electrons will pass through to the detector, and some not. These areas are represented in greyscale, in proportion this ratio.

Image contrast is thus formed by the local differential ratios of transmitted and scattered electrons, which is determined by its local atomic composition as well as the sample’s local thickness and orientation.

At any given accelerating voltage, atoms with higher atomic masses (z-number) are increasingly likely to scatter beam electrons due to their progressively larger and denser nuclei, as well as their correspondingly large cloud of shell electrons.

With biological specimens consisting exclusively of low atomic weight atoms, insufficient electron scattering results in light, washed-out images. Electron microscopists must then rely on heavy metal staining techniques to artificially introduce contrast within the sample.

Mechanistically, these heavy atom stains will preferentially bind to certain structures in the specimen based on composition (e.g. lipids, proteins, glycogen, etc.), locally enhancing electron scattering and creating darkened regions in micrographs.

While the term ‘’staining’’ carries an intuitive understanding of the effect desired, understanding the relationship between atomic number and electron scattering allows for a deeper understanding of the historical rationale to staining with heavy, high atomic number elements as contrasting agents.

While well-established, staining for electron microscopy comes with a set of considerable practical drawbacks.

Lead citrate and uranyl acetate are both commonly used and are very effective staining agents for electron microscopy. However, they are both highly toxic compounds. In the case of uranyl acetate, the staining solution is also mildly radioactive. It is therefore essential to handle these materials with great care, taking necessary safety precautions. The materials require adequate handling infrastructure and ultimately require proper disposal which can be costly.

Staining samples also results in increased sample preparation times as the samples must take up sufficient degrees of each stain before they are stained once more and left to fully dry.

In addition to the time required, achieving consistent staining levels can be challenging, even for an experienced technician. Beyond under-staining or over-staining, the process can introduce stain artifacts that can affect the accuracy of observations.

With both stains, careful preparation and controlled environments are required to avoid undesirable precipitates of the metal salts. While many guidelines for stain preparation and commercially prepared products aim to improve stain stability, this step can often be determinant of the sample preparation quality, as artefact of precipitates can greatly impact the observation of tissues.

Interest in improving on traditional staining protocols is evidenced by the emergence of alternative stains to circumvent common concerns with the most prevalent dyes. These alternatives include Uranyless, platinum blue, various lanthanide salts, and many others. However, these stains all carry a portion of the drawbacks associated with traditional staining.

In addition to the previously stated considerations, staining protocols require that the small and relatively fragile sample grids be repeatedly handled, which can often lead to damaged samples or otherwise poor grid condition by the time the specimen is loaded into the microscope for imaging.

While heavy metal staining has ingeniously been used to alter local electron scattering ratios in a specimen for many decades, it should be noted that this ratio is also a function of the electron beam’s energy as it reaches the specimen. This energy is given by the accelerating voltage of the electron microscopes beam, i.e. by the EM’s operating voltage.

While unstained organic samples simply cannot scatter beam electrons sufficiently to generate adequate contrast at 80-300kV, i.e. conventional TEM voltages, a gentler beam of lower energy electrons will result in a greater spread of scattering ratios as even low-z atoms can scatter beam electrons in sufficient ratios.

In practical terms, lowering the operating voltage of an electron microscope can produce greatly improved contrasts by enhancing the z-contrast inherent to the sample, rather than relying on the introduction of contrasting heavy element stains.

While this approach to scattering and contrast has not always been historically feasible, instruments operating at significantly lower voltages have been made possible by many technological advances since the inception of electron microscopy.

The LVEM 25E by Delong Instruments has many features developed with biological thin sections in mind. Chiefly, it operates at a nominal acerating voltage of 25kV which creates a contrast that is 10x better than at conventional voltages, while remaining sufficiently powerful for most conventional applications.

Remarkably, the tenfold increase in contrast afforded by Low Voltage Electron Microscopy allows even fully unstained biological specimens to be imaged, granting the LVEM 25E its unmatched flexibility for thin section preparation.

Beyond the theoretical discussion regarding the balance between electron scattering, z-number and operating voltage, these differences are best illustrated.

In the following section, micrographs of a muscle thin section embedded and stained according to three different protocols are presented. The micrographs are obtained from sequential sections of the same resin embedded block with similar structures and sarcomere orientations. The resin block was post-fixed with osmium, sectioned to roughly 80nm and deposited onto bare copper grids with varying post-treatment:

• (A) Without any staining

• (B) Stained with 1% uranyl acetate

• (C) Double stained with 2% uranyl acetate & a commercial lead citrate solution

All three grids were imaged at 25kV on the LVEM 25E compact Transmission Electron Microscope.

Here, the tissue section was not stained. Distinct features of the sarcomeres appear in contrast when imaged at 25kV. The Z-lines are distinctive as well as the H-zones. The striated appearance of the sarcomeres is visible.

Depending on the purpose of imaging, this level of contrast may be adequate for the viewer to garner the desired information from the muscle section, saving the operator both the UA and Pb staining steps, along with the associated considerations.

While the time required for the staining steps themselves can be counted in minutes, the time and resources required to maintain stable non-precipitating staining solutions are avoided, as well as the disposal considerations specific to Pb-containing materials.

The hazard associated to both stains, previously discussed, are avoided and the likelihood of damaging the TEM grids before they can be examined is also greatly reduced.

In addition, the artifacts that can arise from staining or from the precipitation of staining agents can also be excluded from the electron microscopist or pathologist’s interpretation of the micrograph. This simplified sample preparation method is only possible when imaging at low-voltages, such as 25kV.

In this trial, the tissue was stained with 1% UA. The same features appear as from the unstained section; however, the natural contrast is enhanced by the addition of the stain, making some features appear slightly crisper and more distinct from their surroundings. Notably, the striations along the muscle are more clearly defined. These sharp features can make the task of focusing the micrograph easier for the operator.

As with the unstained preparation protocol, this preparation method foregoing lead staining is also possible only when imaging at low-voltages such as that of the LVEM25 E.

In the last test of this series, the tissue is stained with 2% UA, as well as a commercial solution of lead citrate. This approach is a conventional preparation of the muscle tissue when imaged at traditional voltages (e.g. 80kV).

With this preparation, the sample appears generally darker, notably around the Z lines. However, the sarcomere’s striations appear very clearly and focus is easily achieved. Brightness can be increased to similar visual effects as with previous preparations by adjusting camera exposure during image acquisition.

While some will prefer this degree of staining, others may not consider it necessary to assess features in the tissue.

Even in the fully unstained sample, the LVEM 25E’s low operating voltage of 25kV allows the sample’s natural z-contrast to be sufficient for many applications. It is important to note that on an 80kV TEM, this would not have been the case.

In the traditionally stained sample (2% UA + Pb), the overall micrograph is less bright, with the darker Z lines features requiring slightly more careful contrasting to evaluate. However, the micrographs overall brightness can be software adjusted with the instrument’s camera settings, yielding satisfying results.

In the 1% UA sample, the same features are visible with a well-balanced range of lights and darks.

While most pathologists’ staining preference will vary with the tissue observed, low-voltage electron microscopy will often allow additional flexibility in staining protocols. Generally, a milder staining process can be opted for as some staining is useful in discerning sample features and for ease in focusing.

In addition to the many practical concerns, eliminating sample staining can also allow truer representations of the materials’ studied.

For many biological organisms (e.g., proteins, viruses), it should firstly be considered that staining agents can potentially impact the pH and salinity of a sample, thus altering the conformation or dispersion of its species. Reciprocally, many stains must be applied with knowledge of sample pH in order to avoid ranges where precipitation can occur.

Additionally, it should be considered that the stains can mask features of a sample if they are used too heavily or if they are poorly selected to discriminate between adjacent structures.

In many cases, the overall detail and contrast of biological organisms can benefit from reduced stain and lowered voltage.

In their 2023 article, Mrazova et al. present a comparison of staining and operation voltage in cyanobacterium micrographs. The authors offer a comprehensive comparison of different permutations of stain, operating voltage and imaging modes, as well as technical discussions of advantages and drawbacks to each.

The unstained samples imaged using low-voltage (25kV) often produced micrographs comparable to samples that were conventionally stained and imaged at higher voltages (200kV). In some cases, their various features were more clearly distinguished.

The author also notes the reduction of impurities (artifacts from staining chemistries) in the unstained low-voltage micrographs. While artifacts from osmium tetroxide (fixative) were present, the unstained samples did not contain precipitated stain artifacts.

In a 2011 article in the Journal of Histochemistry & Cytochemistry, Bendayan et al. used the LVEM 5 electron microscope to examine stain-free pancreatic secretory zymogen granules at low-voltages.

The low-voltage examination of their sample revealed internal compartmentalization of the granules, which was an ultrastructural feature that had been previously predicted, but until then had been fully masked by conventional staining for higher-voltage electron microscopy. This new approach offered new insight into understanding the vital processes occurring in these regions of compartmentalized pancreatic tissue.

In the context of imaging of biological thin sections, low-voltage electron microscopy has many advantages, such as higher intrinsic contrast, which has the potential to lessen the hazards, costs, and concerns associated with traditional staining required for conventional transmission electron microscopy.

In addition, the LVEM 25E is much more compact than traditional high-voltage instruments. Its low operating voltage, paired with innovative design, allows the microscope to stand at less than 5 feet tall, occupying a ~2.5’x2.5’ floor footprint. The instrument requires no special infrastructure (i.e. water cooling, electromagnetic shielding or anti-vibration) and can be installed in nearly any lab space where a standard wall outlet is available.

Having been validated for clinical use in heart and kidney biopsies, the LVEM 25 series microscopes have demonstrated capabilities specifically related to pathology. From its use in multidisciplinary research labs around the world to local hospital pathology centers, the instrument’s compact size and ease-of-use allow excellent imaging workflow and throughput without the need for costly dedicated imaging facilities or months of user training.

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[2] Bendayan, M., Londono, I., & Paransky, E. (2011). Compartmentalization of pancreatic secretory zymogen granules as revealed by low-voltage transmission electron microscopy. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 59(10), 899–907. https://doi.org/10.1369/0022155411418507

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Marie Beaulieu, B.Eng., has multiple years of experience working in the field of solid characterization, imaging, and microanalysis of nanomaterials ranging from active pharmaceutical ingredients to next-generation catalysts. As part of the Applications team at Delong America she enjoys offering practical expertise to electron microscopists from industry and academia alike.