The medium refractive index
The medium refractive index is the Refractive Index (r.i.) of the specimen embedding medium. Below are some frequently used embedding media with their r.i. listed.
A mismatch between the r.i. of the embedding medium and lens immersion medium (Lens Refractive Index) causes several serious imaging problems, including spherical aberration and geometrical distortion. See also Acquisition Pitfalls. These problem becomes more severe when imaging thick samples.
By matching the refractive index of the lens and sample medium as closely as possible, these distortions can be kept to a minimum. For a water-based embedding medium (r.i. 1.32) this can be done by turning towards the use of a water immersion lens. Switching towards an embedding medium with an r.i. that more closely resembles the lens medium r.i. is also an option. There are also specific lenses on the market that are equipped with a correction option for a r.i. mismatch. Unfortunately, these solutions are not always an option, can be costly, and/or only partially solve the r.i. mismatch-related problems.
Huygens corrects for spherical aberration
In the Huygens Software it is very easy to automatically correct for spherical aberration if the lens and medium r.i. parameters are correctly set. Huygens advanced theoretical PSF model will take the depth-dependent PSF changes into account during the deconvolution process. For some nice images examples, see this webpage.
Both the refractive indices of the lens and embedding medium parameters can be edited in the Microscopy Parameter Editor in Huygens. Advanced parameters are available to account for the distance between the coverslip and image stack.
Examples of embedding media
For the frequently used medium glycerol (75%) the refractive index is 1.44. Pure Polyvinyl alcohol can have 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 with a r.i. of 1.40 is offered by several companies.
The r.i. of Prolonged Gold or Diamond increases during curing, and is having a value of approximate 1.42-1.44 after 24 h, with an optimum at 1.46 (Invitrogen). Aqua Polymount (Polysciences) has a R.I. of 1.454 - 1.460.
A paper from the group of Stefan Hell describes the use of TDE for matching the r.i. with the lens medium, and a publication from Frolikova et al., who imaged large oocytes with 3D STED and used Huygens, mentioned the use of the high refractive index embedding medium AD-MOUNT C (RI 1.518), which is commercially available via ADVI Microscopy.
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 keep in mind that the r.i. of an embedding medium can change over time when it for example hardens.