Sample preparation for SIM imaging

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 a newer equivalent (for a hard-set mounting medium). If using Prolong Gold, be aware of potential shrinkage when it cures.

  • Note: Hard-set mounting mediums will flatten out your sample — see figure below.

  • ⚠️ DO NOT USE Fluoromount G. We have had people try this. It does not work.

  • ⚠️ DO NOT 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.

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Effect of Mounting Media on Sample

Effect of different mounting media on sample — hard-set media flatten the sample relative to aqueous media.

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OMX-SR Oil Effect on PSF

PSF bead test on the OMX-SR showing the effect of immersion oil on point spread function quality.