PSF Measurement – prepare the bead sample

  1. Use 100nm fluosphere beads from invitrogen, F-8800 fluoresce at 560nm, F-8801 emit at 605nm and F-8803 emit at 515nm. They are all 2% solids.
  2. Dilute 1:1000 in ddH2O
  3. Dilute 1st dilution 1:1000 in 100% EtOH
  4. Spread 10µL of the 106 dilution on your favourite shape of coverslip as long as it’s a Number 1.5, let dry completely.
  5. Mount in 100% glycerol with antifade (Glycerol-PPD mounting media), you need about 6-8 µL for an 18mm diameter coverslip, or about 10µL for a 22mm diameter coverslip . Mounting in immersion oil causes fluorophore to leach off the beads. Clean the slide with 100% EtOH prior to using, this prevents background spots in your images due to dust.
  6. Seal with Nail varnish, let dry for >3 hrs in the dark.
  7. Image