The most important goal of any confocal microscopy experiment is to maximize photon collection. Forgetting the basics about how photons travel back to your detector is not an excuse for bad data. In this article, I will show you how to increase your image resolution by understanding the importance of the refractive index in optimizing your experiments. By reviewing a few basic aspects of your setup you can ensure that your time spent at the scope is worth it.
Understanding Refractive Index
We all did it. We all did an experiment in elementary school exploring how images shift when viewed through water. This shift is refraction. Refraction happens when light transitions between two different mediums, such as air and water. The degree of refraction is influenced by several factors, the most significant of which is the speed at which light travels through different mediums. In your elementary school experiment example: light traveled through water slower than it did through the air. The amount of refraction is relative to this speed change.
The degree of refraction is also influenced by the wavelength of the light. Different wavelengths refract differently between mediums (think rainbows). While you have been aware of this phenomenon for decades, you might not appreciate how much your microscopy image can suffer if you don’t optimize for refractive indexes.
When optimizing your set up, you must take into consideration the properties of your specific experiments and how these properties will influence refraction. For example, many confocal experiments involve cells that are largely composed of water. As light leaves these aqueous-rich cells it may pass through any of the following mediums with different refractive indexes: oil, water, plastic/glass, media/mounting-media, and air (see Table 1 for common refractive indexes). The better we match up the refractive indexes of all of these mediums the better our ability to collect a high-resolution image.
Table 1. Refractive indexes for common mediums in microscopy
Mowiol (Mounting Media)
Vectashield (Mounting Media)
Flourmount (Mounting Media)
Fresh Prolong Gold (Mounting Media)
1-day-old Prolong Gold
4-day-old Prolong Gold
Mismatched Refractive Index
So what are the consequences of refractive index mismatches? Simply put, mismatches will refract your photons to an undetectable place causing a loss of information. This results in the following problems:
- Spherical aberration.
- Chromatic aberration.
- Reduced resolution.
- Reduced scan depth in the Z-axis.
- Wasted time and effort.
In older microscopes, it was possible to adjust the image distance to correct for spherical aberration produced by refractive index issues (changing the tube length). However, this adjustment is not typically found on modern commercial confocal microscopes because of their complex nature. Instead, spherical aberrations caused by imperfect coverslip thickness, non-homogeneous specimens, or even temperature changes can sometimes be corrected if your objective has a correction collar.
The most likely conditions that will benefit from correction are high numerical aperture (NA) objectives that have major jumps in the refractive index such as from air-to-glass, or from water-to-coverslip. Although complicated, other types of corrector devices exist commercially such as the InFocus™ Dynamic Optical Focusing System. But in reality, adjusting the hardware is a last-ditch and expensive effort. Instead, it is best to ensure you have optimized your setup first.
Optimizing Your Setup
Every experiment is different and will require a different set of optimized considerations. But if you are planning on using a high NA objective (above 40x magnification) and want to visualize a deep structure, or are challenged by weak resolution, check through this list to optimize your set-up:
1) Know Your Objective
Your objective has a wealth of information on it, including:
- the optimal coverslip thickness;
- whether it is a water or oil immersion lens;
- if the objective has a correction collar.
Your first step is to listen to your objective’s advice. This includes making sure to use the correct oil for the lens as not all oils are the same. So consult the manufacturer and spend the money on the one they recommend. Also, keep in mind that temperature matters: heating oil may change its refractive index, and heating water may make it evaporate. If you have access to an upright confocal consider a dipping lens – it can make your life easier by eliminating the need for a coverslip in live imaging.
2) Select Appropriate Coverslips/Chambers
Slight variations in coverslip thickness can make a big difference. Many objectives are engraved with an optimal coverslip thickness of 0.17 mm. However, due to the fact that your cells/specimens are not flush with the coverslip, you must account for this distance. Thus most use a coverslip thickness of 0.13-.016 mm. (When in doubt consult the technical rep for your ‘scope.)
However, even if you purchase a coverslip of proper thickness, slight thickness variations can still occur which can significantly affect your image. Make sure you always buy high-quality coverslips and if your objective has a correction collar, use it.
As with coverslips, it is important to ensure that chambers used for live imaging are optimized for your objectives. In these cases your cells ARE typically flush with the glass/plastic, thus the thickness of the glass is more optimally at 0.17 mm. Plastic chambers are made with a refractive index similar to glass are also made. And remember again that troubleshooting correction collars, different objectives, and different dishes may be necessary.
3) Choose the Right Mounting Medium
Inevitably, the mounting medium lies between your fixed specimens and the coverslip. It is important to choose an optimal mounting medium for your specimen and fluorophores (see Table 1). It is also important to make sure that it is fresh enough to work as intended and that you have taken into account the time after mounting, as many mounting mediums will change their refractive index over time (curing).
4) Adjust for Specimen Heterogeneity
Even after attempting to optimize your objective, coverslip, and mounting media you may still have problems. Inevitably you will meet with variability in your sample that will throw off your resolution: bubbles in the mounting medium, variation in the coverslip thickness, variation in the depth of the mounting medium, or accidental smearing of nail polish over the perfect set of cells. The best way to deal with these problems is to minimize them in the first place – use good techniques and try to correct unavoidable problems with a correction collar.
We hope this article was useful and helped you in understanding the importance of the refractive index in your experiments. If capturing better confocal images is your goal, make sure to check out our top 7 tips for improving your confocal images.
Originally published January 24, 2015. Reviewed and updated February 2021.