Low Voltage Electron Microscopy for Immunohistochemistry:

Immunohistochemistry exploits antibody-antigen interactions to identify specific proteins and other antigens in cells & tissue.

In immuno-electron microscopy, antibody-antigen interactions are exploited by pairing select antibodies with inorganic nanoparticles which are visible under electron microscopy. These antibody-label conjugates are available commercially or can be custom-built solutions.

In preparation for immuno-EM, a processed histological sample (i.e. thin section) is incubated with a primary antibody selected to bind to a specific protein or cell component of interest. Concurrently, an inorganic nanoparticle is attached to a secondary antibody. The nanoparticle conjugated antibody is then incubated with the sample where it binds to its primary antibody-antigen sites.

As a result, the sample antigen is indirectly labelled and roughly located by the most often metallic inorganic particle which appears as a dark, dense deposit when the sample is observed by means of an electron microscope. This immunolabeling process is illustrated in Figure 1.

Where gold particles are employed as the inorganic labels, the technique is referred to as immunogold labeling or immunogold staining.

Since immunogold labeling was first developed, many other derived techniques have been developed to highlight multiple targets concurrently. These rely on strategically selecting the inorganic particles to have distinguishable characteristics such as particle size (e.g., 5 vs. 15nm nanospheres), particle shape (e.g., nanocubes, nanorods) and/or particle species (e.g., silver, palladium). In Figure 3, representative micrographs of various nanoparticle morphologies are presented.

In this way, nanoparticles can be differentiated either visually, according to their size and morphology or, alternatively, by their respective atomic compositions.

Elemental atomic composition can be determined using Energy Dispersive Spectroscopy (EDS), which is a microanalysis technique available in electron microscopes outfitted with associated detectors such as the LVEM 25E which uses the XFlash® 630 detector from Bruker.

In Energy Dispersive Spectroscopy, the sample is imaged while characteristic X-rays resulting from the sample’s excitation by the microscope’s electron beam are collected by an additional dedicated detector.

With each atomic element having a different fingerprint of caracteristic X-ray energies, the obtained spectra can be analyzed to generate a compostional map of the micrograph.

By reliably identifying the inorganic nanoparticle compositions, the acquired EDS data can help to confirm the identity of the targeted antibody regions.

Low-voltage EM offers intrinsically higher contrasts

While many types of electron microscopes can be used for general histological and immunolabeling usage, the LVEM 25E is highly suited for this purpose.

Particularly, the lower acceleration voltage of the LVEM 25E provides intrinsically higher contrast in low atomic-weight materials, such as the ultrastructure found in biological specimens. This allows the possibility for ultrastructure to be visualized without heavy metal stains.

At 25keV, inorganic labels are highly contrasted against the ultrastructure whether stained or unstained. The increased contrast can enhance confidence in identification of the labelled regions as well as their structural features.

The compact LVEM 25E: All-in-One EM

While there are indeed many advantages to low voltage TEM operation in the context of histology, the LVEM 25E all-in-one design allows for further investigation of immunolabeled samples in multiple imaging and analysis modes.

In the example illustrated in Figure 4, immunogold labeling is used to identify insulin in zebrafish beta-cell granules.

In the presented micrographs, the gold particles appear much darker than the ultrastructure in both transmission (TEM) and scanning transmission (STEM) modes. With increasing atomic number, electrons are scattered at higher rates. The dense label particles will scatter more electrons that cross their path than the surrounding organic tissue. As scattered electrons do not reach the S/TEM detector, the gold particles consequently appear as dark spots against the bright organic background in the resulting transmission micrographs.

Using the instrument’s back-scattered electron SEM mode, the gold particles’ distinct bright appearance is consistent with their high-Z composition. Here electrons are back-scattered from the sample and are recorded on the electron source-side of the sample. With increasing atomic number, electrons back-scatter at higher rates. Thus, the gold particles appear as bright white spots against the darker biological field in the resulting micrograph.

Finally, in the LVEM 25E’s EDS mode, the deposits are determinately identified as gold (Au) by processing the EDS spectrum with Esprit® software provided by Bruker.

The LVEM 25 series has been involved in a multitude of fascinating studies, including the study of the regulatory mechanism that underly hypoxic brain injuries.

For example, in their 2022 article in Cells, Stoyanova et al. use immunohistochemistry as one of many techniques employed to further understand the regulatory relationship between Ghrelin and neurogenic and mitotic factors in the hypoxic brain. [4]

Adapted from the aforementioned article, Figure 6 presents TEM micrographs acquired with the LVEM 25 which highlight the comparative intracellular distribution of GHSR1-immunogold precipitates in normoxic, hypoxic and ghrelin-treated hypoxic cerebral cortex cells.

As per the authors, the locations of the precipitates are differentiated by the blue and red arrows, with blue arrows indicating immunogold precipitates found within the cytoplasm, on vesicles and on the plasma membrane. Red arrows indicate nuclear GHSR1-immunogold precipitates.

The LVEM 25E has demonstrated capabilities in life and material science research. From its use in biotechnology research labs to hospital pathology centers, the instrument’s small footprint and ease-of-use increase the ability to rapidly analyze samples without the need for costly dedicated imaging facilities.

In the context of biological thin sections, low voltage electron microscopy has many advantages such as higher intrinsic contrast, which not only offers well-balanced micrographs, but may also reduce or eliminate the need for traditional heavy metal stains such as lead and uranyl acetate.

In immune-electron microscopy, this enhanced contrast and ease of use can improve the process of imaging immune-labelled specimens. The option for elemental mapping can decrease interpretation time while increasing confidence.

[1] Hausrat, T. J., Janiesch, P. C., Breiden, P., Lutz, D., Hoffmeister-Ullerich, S., Hermans-Borgmeyer, I., Failla, A. V., & Kneussel, M. (2022). Disruption of tubulin-alpha4a polyglutamylation prevents aggregation of hyper-phosphorylated tau and microglia activation in mice. Nature communications, 13(1), 4192. https://doi.org/10.1038/s41467-022-31776-5

[2] Meyer, D. A., Oliver, J. A., & Albrecht, R. M. (2010). Colloidal palladium particles of different shapes for electron microscopy labeling. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada, 16(1), 33–42. https://doi.org/10.1017/S1431927609991188

[3] Philimonenko, V.V., Philimonenko, A.A., Šloufová, I. et al. Simultaneous detection of multiple targets for ultrastructural immunocytochemistry. Histochem Cell Biol 141, 229–239 (2014). https://doi.org/10.1007/s00418-013-1178-6

[4] Stoyanova, I. I., Klymenko, A., Willms, J., Doeppner, T. R., Tonchev, A. B., & Lutz, D. (2022). Ghrelin Regulates Expression of the Transcription Factor Pax6 in Hypoxic Brain Progenitor Cells and Neurons. Cells, 11(5), 782. https://doi.org/10.3390/cells11050782

Marie Beaulieu, B.Eng., has many years of experience working with materials ranging from pharmaceuticals to engineered nanomaterials. As part of the Applications team at Delong America she enjoys supporting LVEM users from industry and academia alike.