The medium refractive index

The medium refractive index is the Refractive Index (r.i.) of the Specimen Embedding Medium.

A mismatch between the refractive index of the lens immersion medium (Lens Refractive Index) and specimen embedding medium will cause several serious problems: see Acquisition Pitfalls. For low values of r.i. such as 1.32 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

For the frequently used medium glycerol (75%) the refractive index is 1.44. Polyvinyl alcohol has a refractive index 1.52-1.55.

The embedding medium Mowiol is manufactured by companies like Polysciences, Hoechst and Sigma. Mowiol is intended to match the refractive index of immersion oil. Values seem to range between 1.41 and 1.49. Vectashield Mounting Medium, which does not solidify, has a R.I. of 1.45, and VectaShield "Hard set Mounting medium", which does solidify, has a RI of 1.46. Both are from Vector Labs. Fluoromount G (R.I. 1.40, Interchim), and Prolonged Gold. The RI of Prolonged Gold increases during curing, having a value of approximate 1.42 after 24 h, and 1.44 after 48 h (Invitrogen; see also here. Aqua Polymount (Polysciences) has a R.I. of 1.454 - 1.460.

In general, we recommend to review the product data sheet or contact the vendor for specific details on what mounting media to use. Issues like the refractive index and compatibility with fluorescent dyes need to be considered. Also the r.i. of media that harden can change dramatically in time.