Refractive Index

The refractive index (RI) of a material is the factor (denoted by n) by which electromagnetic radiation is slowed down (relative to vacuum) when it travels inside a medium. For fluorescence microscopy, this means that the light coming from the sample travels at different speeds in different media. Light travelling from one medium to another will not only change in speed, but also wavelength and direction. The change of direction caused by a transition between media is called refraction In optical microscopy, important RIs are the Lens Refractive Index and the Medium Refractive Index.
Both are Microscopic Parameters in the Huygens Software. If there is a mismatch between these two indices this will cause several problems like spherical aberration which can be corrected in the Huygens Software.

Lens Refractive Index

The lens refractive index is the Refractive Index of the medium in which the microscope objective is immersed (the Lens Immersion Medium). Objectives are designed to work on particular immersion media, like oil (RI ~ 1.51), water (RI ~ 1.33) or air (RI ~ 1.00). Usually the medium to be used is written on the objective. Be carefull 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.
The lens RI is directly related to its Numerical Aperture.

The lens refractive index is a Microscopic Parameter in the Huygens Software. The Huygens software can retrieve the lens RI from the metadata of the image file, or it can be set manually.

Medium Refractive Index

The medium refractive index is the Refractive Index of the Specimen Embedding Medium. 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).
For low values of RI such as 1.33 for water, the computed PSF for an oil immersion lens is only valid near the coverslip-medium interface due to spherical aberration effects in deeper layers. In such cases we recommend lenses optimized for the refractive index of the medium.

The medium refractive index is a Microscopic Parameter in the Huygens Software. 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.

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. Moreover, compatibility with fluorescent dyes need to be considered when choosing the medium 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)

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

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).