Best Mounting Medium for Confocal Microscopy?

There are many commercial mounting media available on the market, but not all work well for confocal microscopy.

confocal microscope

This article discusses some of the best mounting media for confocal microscopy, characteristics of a good mounting medium, refractive index, and other relevant topics.

Generally, the best mounting media for confocal microscopy are methyl salicylate or wintergreen oil, BABB (Benzyl Alcohol/ Benzyl Benzoate), and TDE (2,2’-Thiodiethanol). Commercial products include Vectashield and Prolong Gold. The right mounting medium can prevent fluorescence fading, preserve the integrity of sample for storage and keep it safe from microorganisms and moisture. Let’s discuss this in detail.

First a brief primer on the basics.

Confocal Microscopy Basics

Also known as confocal laser scanning microscopy, confocal microscopy is an optical imaging technique that creates a 3D picture of a specimen by employing fluorescence and lasers. Thanks to the laser, and upwards of 3 can be used on one slide, it allows collection of very clear images from a thick sample, one thin section at a time. Confocal microscopy has found many applications in biological sciences, including microbiology, genetics, and cell biology. It is particularly helpful in studying live cells.

The mounting medium is the substance that holds the sample in place between the slide and the coverslip. It allows for long storage of the sample and protects it from damage. Refractive index plays a vital role in selecting a mounting media. The mounting media, coverslip, sample, and glass should have a refractive index as close as possible to each other.

Here is a useful video on confocal microscopy basics:

Best Mounting Media For Confocal Microscopy

Here are some of the best mounting media for confocal microscopy:

1. Methyl salicylate or Wintergreen oil

Wintergreen oil is a natural product that is extracted from the leaves of the wintergreen plant. Methyl salicylate is the active ingredient of this oil. According to research carried out at The National University of Malaysia, wintergreen oil is more environmentally friendly and safer than the solvent xylene for mounting specimens on slides. Xylene is a colorless chemical that is used as a clearing agent in the medical laboratory.

2. BABB (Benzyl Alcohol/ Benzyl Benzoate)

This solution consists of one part benzyl alcohol and two parts benzyl benzoate. It has been used as an optical clearing agent for many years. While BABB clears tissues very fast, it quenches fluorescent proteins. Fluorescence quenching is a physiochemical process that decreases the fluorescent intensity of a specimen. Also, the solution can be applied only after dehydration of tissues because it is not miscible with water.

Methyl salicylate and BABB are the best mounting media when imaging depth is critical.

3. TDE (2,2’-Thiodiethanol)

Also known as thiodiglycol, TDE is an organosulfur substance that is mixable with organic solvents and water. And it is the best mounting medium when maintenance of cell morphology is important.

Commercial Mounting Media for Confocal Microscopy

1. Vectashield

Vectashield has been used for confocal mounting for many years, I have used it extensively and it has good anti fading properties. It comes in several versions- one that does not solidify and can be used with tissue that needs to be readily removed, and sealed on the perimeter with a roll of putty or nail polish, and one that solidifies and does not require any other sealants.

2. Vectashield Vibrance

This is a newer version of the popular commercial mounting medium which is hard setting and therefore does not require nail polish to close the slide. It also has improved anti fade qualities and no background, giving a more vibrant, stronger staining signal.

3. Prolong Gold

Prolong is another very good confocal mounting medium I have experience with, which has antifade qualities and a good refrative index of 1.47. It is also available with DNA stain built in if desired.

What Are The Characteristics Of A Good Mountant?

The refractive index of the mounting medium should be near 1.518.
It should be resistant to the growth of bacteria.
It should not change in pH or color
It should not cause distortion.
It should not dissolve out.
It should not wash away any stain.
It should mix easily with toluene and xylene.
It should stay stable once set.

Why A Mounting Medium Is Important?

Here is why the mounting medium is extremely important:

It enhances the quality of the image
It supports and stabilizes the sample.
The mounting medium keeps the specimen safe from external contamination, such as moisture and bacteria.
The refractive index of the mountant can have a notable impact on the quality of the picture.

Fluorescence Mounting Media Requirements

Fluorescence mounting media should have a high refractive index
It should buffer fluorophores (synthetic polymers, organic compounds, or proteins) and the specimen.
It should prevent photobleaching. Also called fading, photobleaching happens when a dye loses its ability to fluoresce owing to photochemical alteration.

Recipe for Fluorescence mounting media

20mM Tris, pH 8.0.
5% N-propyl gallate. It is an ester formed by a condensation reaction between propanol and gallic acid. It helps prevent fading.
50-90% Glycerol. Glycerol increases the refractive index of the mounting media, and a higher refractive index leads to greater resolution. The fluorescence image gets better as the concentration of glycerol gets higher.
Heat the solution to 37°C and vortex.
Just 6-8ul of mounting media should be used for every 18mm coverslip.
Use nail polish to seal the coverslip.
Mounting media should be stored at 4°C.

DIY Mounting Media

Here is how to prepare mounting media yourself:

Prepare mounting medium by mixing one part PBS with nine parts of glycerol.
Adjust the pH between 8.5 and 9.0. This pH is ideal in preventing the quenching of fluorescein.
You can also add an antiquench agent, such as ascorbic acid, propyl gallate, and p-phenylenediamine, to the mounting media to slow the fluorescence quenching.

Confocal Microscope And Widefield Fluorescence Microscope

Widefield microscopy is an imaging technique that allows the localization and detection of proteins and cells. In this technique, the complete specimen is illuminated with light of a particular wavelength, exciting fluorescent cellular objects within it. The fluorescence emitted by the specimen is either captured by a camera or seen through an eyepiece.

The maintenance cost of a widefield microscope is low compared to a confocal microscope. A widefield microscope is considerably cheaper than a confocal microscope because of its less complicated optics and the sensitivity of its detectors. It allows imaging of alive or small biological samples. However, a widefield microscope is not good for examining thick specimens or specimens that scatter light significantly. Imaging such specimens with this microscope would give vague images with high background.

On the other hand, a confocal microscope enables to get 3D images of the specimen with high-quality resolution. It is good for examining thicker structures or ones that scatter light. Confocal microscopy offers more resolution in-depth and reduces background fluorescence away from the focal plane.

Refraction

Refraction is the change of direction of light when it passes from one medium into another medium of different densities.

Also known as the index of refraction, the refractive index is the ratio of the speed of light in a particular medium to its speed in a vacuum.

Below is a list of refractive indices for common mediums in microscopy –

Glass – 1.52

Immersion Oil – 1.51

Glycerol – 1.47

Water – 1.33

The refractive index is a vital parameter in calculating numerical aperture in light microscopy. A numerical aperture is a dimensionless number describing the light gathering capability of a condenser or objective. It is an essential factor when trying to distinguish detail in a sample viewed down the microscope.

Matching the refractive index of your mounting medium to the refractive index of the glass components is essential as it will optimize the brightness and resolution of the images.

Mismatched refractive indices, on the other hand, can cause chromatic aberration, decreased resolution, or spherical aberration, which can affect the brightness of the specimen.

Tips To Reduce Photobleaching

Here are some useful tips to reduce photobleaching.

Use resistant dyes

One of the effective ways to reduce photobleaching is by using dyes that are less vulnerable to bleaching. Janelia Dyes, AttoDyes, Cyanine Dyes, DyLight Fluors, and Alexa Fluors are all dyes that are resistant to bleaching.

Decrease light intensity and time exposure

Photobleaching can also be reduced by reducing light intensity exposure to fluorophores. Decreasing light intensity also decreases the number of excitation-emission cycles and extends the life of fluorescent molecules. However, it’s important to note that decreasing light intensity can decrease emission signals. Hence, you need to find a balance so that the quality of the image is maintained. Additionally, decreasing time exposure to light sources decreases the number of excitation-emission cycles and decreases photobleaching.

Keep in Mind

A mounting medium plays a vital role in enhancing the clarity of an image during confocal microscopy. It is also essential to protect your sample from physical damage. And the refractive index plays a crucial role in selecting a mounting medium. The refractive index of mounting media, coverslip, sample, and glass should be as close as possible to each other.

Here is a useful video about mounting media:

I hope this article has helped you decide on a mounting medium for confocal microscopy.

Click the following link to learn how to perform deconvolution.