Structured Illumination (SIM) imaging is extremely sensitive to spherical aberration. To get the highest quality images (or even to get proper reconstruction), you need to minimize spherical aberration. This means:
You should use high precision 0.17 mm coverslips
You should mount in an appropriate mounting medium. Nikon recommends mounting either in 2,2-thiodiethanol or in Prolong Gold or newer equivalent (for a hard-set mounting medium). If using Prolong Gold, be aware of potential shrinkage when it cures.
Note: Hardset mounting mediums will flatten out your sample! See figre below.
DO NOT USE Fluoromount G. We have had people try this. It does not work.
DO NOT USE use mounting medium which contains DAPI or other fluorophores.
- You should have your sample as close to the coverslip as possible (e.g. cells grown on coverslip not on slide).
- you should use bright and stable fluorophores.
Mounting Media effect on sample
For OMX-SR Oil effect on PSF
