Looking at the big wide world of cells through electron microscopy: A journey in 2D and 3D

If you are a fan of action flicks, you probably have seen at least one movie in 3D. The 2D (via transmission EM) and the 3D (via scanning EM) world of cells, seen through the evolving tool of electron microscopy (EM), tell their own stories. TEM transmits a beam of electrons through a very thin sample, to image primarily the interior of cells and tissues. SEM scans a beam of electrons across the surface of a sample to image the surface topography.

SEM recreates the cellular world in 3D, showing us the compound eye of a fly, or close-up images of cells interacting with each another. Ever since the 1940s, when scientists obtained unprecedented resolution of chicken fibroblasts by TEM—and ushered in the era of cell biology—laboratories have been testing the boundaries of EM. The specialized microscopes have been used to magnify the face of a mosquito (SEM), to show two astrocytes connecting at the same synapse (TEM), and to identify promising drug targets (TEM).

More recent innovations of EM include cryo-EM, which earned three pioneering scientists a Nobel Prize for developing a new way to assemble precise 3D images of biological molecules like proteins, DNA and RNA. Other creative uses of EM include immuno-EM and ultrastructural morphometry and stereology. Immuno-EM specifically localizes antigenic targets at the ultrastructural level with nano-gold particles that can range from 6 nm to 25 nm. Morphometric or stereological analysis can be used to learn more about the morphology of tissues at an ultrastructural level.

For example, morphometric analysis can be applied to measure or quantify cellular organelles, inclusions, viral particles, or nanoparticles imaged by TEM. Uniquely optimized image analysis algorithms can be created and applied to electron micrographs to identify and measure or count the features of interest. Marrying quantitative pathology with immunohistochemistry through immuno-EM allows us to identify and quantify particular antigenic targets at the subcellular level. In addition, tissues can be sampled according to stereological principles using systematic uniform random sampling (SURS), and resulting electron micrographs can be analyzed to estimate volume, number, length, or surface area of objects within a defined region of interest.

So what’s not to like? The main drawback to EM is that it is both expensive and time-consuming when compared with routine histology techniques. Also, the equipment and expertise are typically localized to universities, pharmaceutical companies, and clinical research organizations. Yet EM, which had gone through a downturn in the 1980s because of the advent of new immunohistochemical, cytogenic, and molecular biology techniques, has rebounded in the last 10 years or so. Reasons for the renewed interest in EM include the realization that these techniques do not resolve all diagnostic problems and the growth of nanotechnology, which deals with nanoscale particles or structures with dimensions less than 100 nanometers.

And while EM still tends to be a tool associated with academic research, it can be performed in compliance with GLP guidelines. In fact, CROs and other commercial laboratories are using the technique routinely to help assess new drug candidates.  These tools help clients investigate drug-related ultrastructural changes in cells and tissues that are unresolved by light microscopy. Our lab has even characterized the ultrastructure of nanoparticles, proteins, peptides, phages, and viruses.

Other novel imaging modalities in the drug development space include Time-of-Flight secondary ion mass spectrometry, matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (MALDI-IMS), magnetic resonance imaging (MRI), and ultrasound.

Once a novelty, different innovations of EM are now used throughout the drug development process—from discovery to clinical safety assessment—and its utility continues to grow and adapt with technological advances and will remain applicable for years to come.

Who knows what tomorrow’s iteration will look like?

Amera Remick, Executive Director of Pathology Associates for Charles River, is presenting a talk March 14 on innovations in EM at the Society of Toxicology’s annual meeting in San Antonio, Texas. Al Inman, Manager of Electron Microscopy at Charles River Pathology Associates, and Danielle Brown, a Principal Veterinary Pathologist at Charles River, contributed to this blog post.