Editorial Feature

Advancements in Immunolabeling for Electron Microscopy

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For biological imaging purposes, electron microscopy (EM) is a superior imaging technique that has been shown to achieve the highest level of spatial resolution in protein localization as compared to any other form of cellular imaging. While this may be true, current EM techniques often lack the ability to label specific proteins as a result of the inability of immunolabeling antibodies to withstand the strong fixation required for EM samples.

Challenges for Immunolabeling in EM

Currently, the only target proteins that can be labeled and visualized by EM are those which are present at cut tissue surfaces. To achieve this level of specific labeling, bulky gold particles can be used to replace eosin staining, thereby promoting the catalytic amplification of the proteins through photooxidation of diaminobenzidine (DAB). Even when this staining technique is used, the eosin-conjugated macromolecules remain limited in their diffusibility into the cell membrane and typically require the addition of a detergent in order to allow for the stain to permeabilize cells.

Researchers have also investigated the potential incorporation of genetic labeling methods with EM; however, there is currently no genetically encoded tag for EM contrast that can mimic what is achieved in fluorescent light microscopy imaging. The development of a high-affinity and highly selective antibody capable of recognizing cross-links in EM samples is therefore highly desired within biomedical research.

Advantages of Immuno-EM

The use of immunofluorescence microscopy (IF) is a widely popular technique for the analysis of cell and tissue samples; however, immuno-EM (iEM) is capable of providing a wide range of advantages as well. For example, immunogold labeling in iEM provides a greater amount of detail on cellular structures as compared to IF methods. Furthermore, quantitative analysis of immunogold-labeled samples is easier to count and quantify as compared to IF.

Correlative Light-Electron Microscopy (CLEM) and Immuno-Electron Microscopy

Correlative light-electron microscopy (CLEM) is a powerful imaging technique that combines the advantages associated with both light microscopy (LM), or fluorescence microscopy (FM), and EM. CLEM combines the ability of LM to capture wide-field images of the whole cell with the high resolution of EM, thereby allowing researchers to visualize specific cellular structures and processes of interest with EM and simultaneously capture the whole cell. While useful in theory, the technical sophistication of CLEM equipment can be challenging for many scientists and is typically only applicable to cultured cell samples.

To overcome these limitations of CLEM and target specific macromolecules within a sample, a recent study discussed the development of a novel CLEM method used to analyze tissue samples. To this end, the researchers utilized optimal cutting temperature (OCT), which is a commonly used tissue and cell embedding medium for FM purposes. OCT-embedded cryostat samples are capable of maintaining sample preservation for extended periods of time while stored in -80°C, which prevents the need for researchers to continuously generate fresh specimens when alternating between microscopy techniques.

To test their CLEM methodology, the researchers examined the localization of rhodopsin, which is a pigment present in the rods of the retina, within induced pluripotent stem cell (iPSC) derived optic cups, which is a cited in vitro model used to study retinal tissue physiology and disease. Following the preparation of the OCT cryostat sections, tissue sections were permeabilized and labeled with anti-rhodopsin antibodies for FM analysis. For EM analysis, the cryostat sections were re-fixed with Nanogold or FluoroNanogold antibodies.

Although the research in this study did not use a correlative imaging approach, FM provided a visualization of the localization of rhodopsin and phagosomes, whereas EM was used to provide information on the precise position of these macromolecules within the samples. The researchers are hopeful that their embedding methodology, which incorporates commonly used embedding techniques and is cost-effective, can be applied for both CLEM and iEM of cryostat sections.

Sources and Further Reading

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Benedette Cuffari

Written by

Benedette Cuffari

After completing her Bachelor of Science in Toxicology with two minors in Spanish and Chemistry in 2016, Benedette continued her studies to complete her Master of Science in Toxicology in May of 2018. During graduate school, Benedette investigated the dermatotoxicity of mechlorethamine and bendamustine, which are two nitrogen mustard alkylating agents that are currently used in anticancer therapy.

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