‘Staining’ of Biological Samples when Using Electron Microscopy: Why stain density matters

By Dr. Liji Thomas, MDReviewed by Georgios Christofidis, Ph.D.

When samples are viewed under the light of a microscope, details can be viewed more clearly using light-absorbing stains.

With transmission electron microscopy (TEM), heavy metals are used to ‘stain’ biological tissues by absorbing or scattering some of the electrons that would otherwise be projected onto the microscope’s lens.

The scattering offers a contrast between the different structures, which is important given the almost transparent nature of many biological tissues to electrons.

Without such staining, the low contrast of a biological sample is an important limitation on the resolution of fine details on an ultrathin section that can be achieved using the electron microscope.

Increasing tissue contrast by staining

Such contrast is proportional to the difference in the mass per unit area of the tissue organelle or macromolecule being visualized and the surrounding embedding material that remains after being under the electron beam. To gain adequate contrast, complete removal of the embedding material is required, which will cause undue distortion of the material. The other option is to increase tissue density by using heavy metals.

However, post-embedding staining is preferable to optimize the level of contrast while minimizing distortion for more delicate structures.

Other advantages of staining the embedded section include the greater uniformity of staining, and the ability to compare serial sections of the same tissue block which have been treated with different methods of staining.

These heavy metal atoms attach to macromolecules or organelles within the tissue sections, making them more electron-dense, so they look dark against a lighter background.

For instance, phosphate and amino groups in the biological samples attach strongly with uranyl ions and lead ions to negatively charged elements.

The importance of stain density

Stain density is important in obtaining the best quality of data from electron microscopy using several newer techniques, such as serial block-face electron microscopy (SEBM), or focused ion beam scanning electron microscopy (FIB-SEM).

A lot of experimental work is wasted when specimens which are not stained following the imaging procedure, are visualized under the electron microscope, yielding images of lower quality.

Techniques such as SBEM and FIB-SEM have helped to provide three-dimensional ultrastructural details at the nanoscale which can help to elucidate the mechanisms for an array of important cellular processes.

Both of these depend on the imaging of cells or tissues embedded within a medium and subjected to scanning electron microscopy (SEM), where electrons are backscattered from a heavy metal stain incorporated into the fixed biological specimen.

With these techniques, the heavy metal stain must be introduced before data acquisition because the block cannot be post-stained. The best results are obtained only from an optimized sample because scanty staining causes low signal-to-noise ratios which makes the ultrastructure less visible.

This is because too little stain causes the block to become charged, distorting the stacked images and rendering them useless.

On the other hand, overstaining will cause obscuring of the ultrastructure. The flow of electrons on the block should be 20 electrons per nm², because excessive radiation can cause the block to shrink, causing uneven cutting when the cutting increments are reduced.

How is stain density measured?

Staining parameters are important and can be determined by methods such as the collection of annular dark-field images in the scanning transmission electron microscope (STEM) at different angles.

This provides elastically scattered electrons with different angular distributions, depending on whether they interact with light or heavy atoms. This gives a quantitative measure of how heavy atoms (stain) are distributed among the light atoms (sample and embedding resin).

TEM imaging and stain density estimation

Another technique involves the use of bright-field TEM to image sections cut from the same block that is to be examined by SBEM or FIB-SEM. This method is more versatile for slice thickness and radiation-induced mass loss, and is also rapid, taking only a few minutes. In most cases, the staining is performed using osmium, lead, and uranium. Among these, lead is present at the highest levels.

TEM imaging is suitable for finding the stain concentration in any other mode of electron microscope imaging too, requiring only the measurement of the thickness of the slice and the elastic scattering cross sections for the heavy atoms used for staining, mostly osmium, lead, and uranium.

Once the specimen is fixed, embedded and stained en bloc, imaging is done to calculate the transmitted intensity through the measured thickness of the section that contains embedded stained cells.

The thickness is determined by the microtome settings or by doing low-dose electron tomography of the sections containing deposited nanoparticles that serve as fiducial markers. These nanoparticles are used as a point of reference within the field of view of the imaging system.

The stain concentration is derived by dividing the slope by the average elastic cross-section for the heavy metal stain used.

The partial elastic scattering cross-section of lead is used because it is the most abundant element and lies between osmium and uranium. Such cross-sections are available from standard reference databases.

Advantages

The measurement required to find the concentration of the stain using the TEM-based technique is a dimensionless one and is therefore independent of the light atoms, making the method a very reliable one even if the mass is lost during bright-field imaging or if the section is contaminated during TEM imaging.

The measurements are achieved just as easily with beam energies of 100 or 120 keV as with a 300 keV TEM.

Moreover, the rapidity of the method and the convenience of having to simply record a few bright-field images to get the measurements, make it an invaluable method to estimate stain density.

Further Reading

• All Microscopy Content
• What is Microscopy?
• Microscopy and Artificial Intelligence
• Scanning Electron Microscopy (SEM): An Overview
• Investigating the Intersection between Microscopy and Healthcare