The End of the Line: Considerations for Mounting Media Selection

So now you’re probably asking: How do I choose the right mounting medium? Let’s go over some of the basics …

Water-Based vs Solvent-Based

Mounting media generally fall into two categories: water (aqueous)-based, or solvent (non-aqueous)-based liquids. Water-based mounting media allow you to mount directly from aqueous buffers and provide a suitable pH for sample preservation, as well as the viscosity needed to easily apply a glass coverslip. Within this category, there are two subsets. Non-setting, water-based media stay in a liquid state, which means you may need to use a sealing material, such as nail polish or paraffin wax, around the perimeter of the coverslip to prevent motion. The advantage of a non-setting medium is that the refractive index is stable at all time points (don’t worry, we’ll explain later) and microscopic viewing can occur as soon as the sample is prepared. Setting, water-based media, as you might imagine, form a solid film upon drying (generally, within 1-4 hours) and do not require any extra sealant. The refractive index, however, may require up to 24 hours to completely stabilize.

The use of solvent-based, or resinous, mounting media goes back to the 1830s, when Canada balsam, a resin made from a balsam fir tree, was first used. The vast majority of solvent-based mounting media are setting and will solidify with time, sealing the coverslip and the sample. Generally referred to as permanent mounting media, they will preserve and protect the sample with an optically clear film for years. However, when using these mounting media, it is important to consider that the samples cannot be mounted directly from aqueous buffers since the mounting medium is not miscible with water. In most cases, the slide must be transferred through a series of dehydration steps, removing the water from the specimen and converting the sample’s environment to a non-aqueous one. Usually, a series of dehydration (ethanol) and clearing (xylene) solutions are used to accomplish this.

Refractive Index

At the most basic level, any aqueous buffer could serve as a mounting medium. However, the refractive index (RI) of water (1.33) differs significantly from that of the glass slide and coverslip (1.51), oil-immersion liquids (1.51), and the tissue section itself (1.38–1.46) (2). Because of this, using water as a mounting medium can result in RI mismatching. With mismatching RIs, focal point broadening, or spherical aberration occurs, which can cause resolution degradation and affect sample brightness (3). That’s why matching the RI of your mounting medium to the RI of the glass components (and immersion liquid, when used) will optimize the clarity, resolution, and brightness of the images.

Many water-based mounting media use glycerol as the major component since the RI of glycerol (1.47) is close enough to glass to allow high quality imaging, while the final dried film of a solvent-based mounting media will generally have an RI of 1.45–1.49, which also allows for high-quality imaging.

Technique For Mounting Tissue Sections

It can take a bit of practice to prepare mounted slides without any air bubbles, but, generally, there are two methods for coverslipping tissue sections: the slide method and the coverslip method. In the slide method, you apply a small amount of the mounting medium onto the tissue section, place one end of the coverslip on the slide, slowly lowering the coverslip with the aid of a dissecting needle or forceps, and then allow the slide to air dry in a horizontal position. Going slowly is the key to this method as it makes air bubbles less likely to form. In the coverslip method, rather than adding the mounting medium directly onto the tissue, you instead apply it to the center of the coverslip. You then slowly lower the inverted slide until you make contact with the coverslip, letting the surface tension pull the coverslip up, invert the slide quickly, and allow it to air dry in a horizontal position.