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 No 1.7 high-precision (0.17 +/- 0.005 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.

Keep in mind what mounting media you will be using and what it does to your sample

 

Your Point Spres Function (PSF) and Oil will be important steps! this is why your high-precision coverslips and mounting media are critical!

 

 

NOTE: for our OMX, a preparation you will need to have in mind! is getting your data off

  • Can I use Box/drop box?
    • NO-As the OMX has gotten older, the Operating system is no longer compatible with versions of browsers that can run box and dropbox
  • How does my hard drive need to be formatted?
    • FAT32 or NTFS. These are the ONLY 2 that will work
    • Please Note FAT 32 can not handle file sizes larger than 4GB
    • NTFS has limited Read-only capabilities on Mac.