Refractive Index
The refractive index (RI) is a physical property of a material and has many implications for light and fluorescence microscopy. The RI determines how light changes in direction, speed and wavelength when light crosses from one medium into the next. We can consider the microscope as a continuous optical path consisting of different materials with their respective RIs, all causing slight alterations in how the light travels through the microscope. The change of direction caused by a transition between media is called refraction.
A refractive index mismatch is a common image acquistion pitfall, more information can be found at the bottom of this page.
Definition
The refractive index of a material is the factor (denoted by n) by which electromagnetic radiation (light) is slowed down (relative to vacuum) when it travels inside a medium.A refractive index mismatch is a common image acquistion pitfall, more information can be found at the bottom of this page.
Figure description: Schematic representation of the refraction of light as it travels from a medium with a low refractive index (air) to a medium with a higher refractive index (water).
Microscope embedding media
In fluorescence microscopy, the refractive indices of the lens immersion medium and the sample embedding medium used for image acquisition should match to get the best image quality.In the Huygens Software
Throughout the Huygens Software, the refractive indices of the lens immersion medium (lens refractive index) and sample embedding medium (medium refractive index) are microscopic parameters. To correctly estimate the PSF needed for deconvolution, it is important that the refractive indices of the embedding media are set correctly for each image. Huygens can retrieve both refractive indices from the metadata but usually the embedding medium is not stored. Therefore it is advised to check these values manually inside the microscopic parameter window. If the lens and medium RI is correctly set, the theoretical PSF model in Huygens will take the depth-dependent PSF changes due to spherical aberration into account with deconvolution.Figure description: Schematic representation of the refraction of light as it passes through the sample embedding medium and lens immersion medium. The sample is simplified to a single red fluorophore. With immersion oil (left panel) the refractive index from sample to objective is mostly consistant, giving no refraction. Imaging through air (right panel) causes strong refraction, resulting in light being bend away from the objective.
Lens Immersion Medium
The lens immersion medium is the medium that is in contact with the objective lens, and is often air (RI ~ 1), water (RI ~ 1.33) or oil (RI ~ 1.51).Light refraction and refractive indices play a crucial role in determining the resolution of the microscope. For increased resolution, the microscope objective needs to have a high Numerical Aperture (NA). The NA is directly related to the resolution following the Rayleigh criterion.
Although the NA is a fixed property of the objective, the effective NA can be increased. This is done by using a different lens immersion medium, that matches the RI of the glass lens. Glass has an RI of 1.5, while air has a refractive index of 1. This means light traveling through air to reach the objective will be refracted and scattered, resulting in less light being focussed by the objective. To overcome this issue, many microscopes use oil immersion (RI ~ 1.51), that matches the RI of glass allowing more light to be captured by the objective.
Objectives are designed to work with particular immersion media, usually the medium to be used is written on the objective. Be careful to always use the correct match between objective and lens immersion medium. Using oil immersion with a water objective could permanently damage the objective! For more information on air lenses (also called dry lenses) see Air Lens Correction.
Sample Embedding Medium
The sample embedding medium is the medium that surrounds the sample. For most biological samples, the medium RI depends on the embedding medium (mounting medium for fixed cells) and the glass coverslip (RI ~ 1.47 - 1.52).To avoid refraction, the sample embedding medium should have a similar RI as the lens immersion medium, as a homogenous RI across the entire sample will limit diffraction and allows fo a greater imaging depth. Live tissue has a RI of 1.36 - 1.38, cultured cells have a RI of approximately 1.4, but note that the RI is highly depended on the type of sample and the cell type. To minimize refraction, it is adviced to choose a embedding medium that matches the RI of your sample as close as possible.
To give your sample a consistent and higher RI, tissue clearing can be used. This is a technique used to remove dehydrating components from the cell and give the entire sample a similar RI. In this process, lipids (RI ~ 1.47) and intra- and extracellular fluids are replaced with the clearing solution that has a higher RI, matching that of proteins (RI ~ 1.5) (Richardson et al., 2021). The homogeneous RI limits refraction and makes the sample more transparant. This technique is used for large (ranging from 100 um to several centimeters), fixed biological samples and is particularly useful for studying whole organisms, organs or organoids (Ariel et al., 2016).
Examples of embedding media
In general, we recommend to review the product data sheet or contact the vendor for specific details on what embedding or mounting media to use. The embedding medium should match the RI of the coverslip and lens immersion medium as close as possible. Moreover, compatibility with fluorescent dyes need to be considered when choosing the sample embedding medium. In addition, mounting media that harden (cure) will change their RI over time and only reach their advertised RI when fully cured.For fixed cell experiments many different embedding media (mounting media) are used. Many of these media are well-suited for prolonged retention of fluorescence. Some examples of mounting media and their RIs are listed below.
Sample embedding media
Glycerol (75%)
Polyvinyl alcohol
Mowiol (Polysciences, Hoechst & Sigma)
Vectashield Mounting Medium (Vector Labs)
VectaShield "Hard set" (Vector Labs)
Fluoromount G (Interchim & Invitrogen)
Prolonged Gold (Invitrogen)
Aqua Polymount (Polysciences)
Glycerol (75%)
Polyvinyl alcohol
Mowiol (Polysciences, Hoechst & Sigma)
Vectashield Mounting Medium (Vector Labs)
VectaShield "Hard set" (Vector Labs)
Fluoromount G (Interchim & Invitrogen)
Prolonged Gold (Invitrogen)
Aqua Polymount (Polysciences)
Refractive index
1.44
1.52 - 1.55
1.41 - 1.49
1.45
1.46
1.40
RI increases during curing, approximately 1.42 after 24 h, and 1.44 after 48 h.
1.454 - 1.460
1.44
1.52 - 1.55
1.41 - 1.49
1.45
1.46
1.40
RI increases during curing, approximately 1.42 after 24 h, and 1.44 after 48 h.
1.454 - 1.460
For live-cell experiments, many of the mounting media are unusuable due to toxicity. However, there are some embedding media that can be used with live cells to match the RI. For example Iodixanol, which is a tunable non-toxic IR matching medium Iodixanol has a RI of 1.333 – 1.429 (see also here).
Refractive Index Mismatch
The lens RI should match the medium RI. A mismatch between the refractive index of the lens immersion medium and the specimen embedding medium will cause several serious problems:1. Spherical Aberration
2. Axial (geometric) distortion: the Fishtank Effect.
3. Reduction of the effective Numerical Aperture by Total Internal Reflection
If you have a refractive mismatch there are two main steps you need to take to correctly restore your image.
1. Correct spherical aberration by using the Huygens theoretical PSF for deconvolution. If you set the mismatch in the microscopic parameters and use a theoretical PSF, this is automatic and you do not have to take any action. If your image is thin (low depth in Z), a measured PSF can also do fine, but the theoretical PSF is advised. (see spherical abberation in Huygens for more information).
2. Correct the geometrical distortion (fishtank effect). Do this by setting the sampling size in the Z direction to correct for the distortion in the microscopic parameters. You can find how to calculate this distortion under fishtank effect
1. Correct spherical aberration by using the Huygens theoretical PSF for deconvolution. If you set the mismatch in the microscopic parameters and use a theoretical PSF, this is automatic and you do not have to take any action. If your image is thin (low depth in Z), a measured PSF can also do fine, but the theoretical PSF is advised. (see spherical abberation in Huygens for more information).
2. Correct the geometrical distortion (fishtank effect). Do this by setting the sampling size in the Z direction to correct for the distortion in the microscopic parameters. You can find how to calculate this distortion under fishtank effect
Mismatch distorts PSF
When imaging with a refractive index mismatch, the PSF will increase with image depth due to spherical aberration. Fortunately, the theoretical PSF in the Huygens Software can correct for the mismatch between indices if this mismatch is correctly specified in the microscopic parameters of the image.A refractive index mismatch is a common problem, see also Acquisition Pitfalls for other common image acquisition issues.
Figure description: Schematic representation of axial distortion (fishtank effect) due to a refractive index mismatch. As the light rays coming from the sample are bend, an apparant object is imaged that is elongated relative to the actual object. The mismatch in RI causes light rays to appear as if they originate form higher depths than they actually are, stretching the objects. Schematic based on Lichtman et al. (2020).
Practical example
To illustrate the effect of spherical aberration on your 3D image consider the following example (explained in more detail for spherical aberration).
The images to the right are obtained with a confocal microscope, imaged with oil (RI = 1.515) as the lens immersion medium and Vectashield (RI = 1.45) as the sample embedding medium. Even this small mismatch causes problems, mostly in the deeper parts of the image. Deconvolving the image gives much better resolved structures, yet they still appear elongated due to the fishtank effect. The mismatch in RI causes spherical aberration, as oblique light rays from a same point source are bend more strongly than central light rays on the interface between the mismatching media. This causes objects to appear blurred and out-of-focus.
Figure description: MIP projections along the XZ axis of 3D Drosophila brain images, without post-acquisiton processing (original, left), deconvolved without spherical aberration correction (middle) and deconvolved with spherical aberration correction (right). Image courtesy of Tory Herman, Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA
Related
RefractionMicroscopic parameters
Spherical Aberration
Total Internal Reflection
Fishtank Effect
References
1. Ariel P. A beginner's guide to tissue clearing. Int J Biochem Cell Biol. 2017 Mar;84:35-39. doi: 10.1016/j.biocel.2016.12.009. Epub 2017 Jan 7. PMID: 28082099; PMCID: PMC5336404.2. Diel, E.E., Lichtman, J.W. & Richardson, D.S. Tutorial: avoiding and correcting sample-induced spherical aberration artifacts in 3D fluorescence microscopy. Nat Protoc 15, 2773–2784 (2020). https://doi.org/10.1038/s41596-020-0360-2
3. Dirckx, J. J. J., Kuypers, L. C., & Decraemer, W. F. (2005). Refractive index of tissue measured with confocal microscopy. Journal of Biomedical Optics, 10(4), 044014. https://doi.org/10.1117/1.1993487
4. Richardson DS, Guan W, Matsumoto K, Pan C, Chung K, Ertürk A, Ueda HR, Lichtman JW. TISSUE CLEARING. Nat Rev Methods Primers. 2021;1(1):84. doi: 10.1038/s43586-021-00080-9. Epub 2021 Dec 16. PMID: 35128463; PMCID: PMC8815095.
Last updated: June 2024