Advanced Topics

Creating a library IRF


  • Measurement of the temporal profile of an HRI for particular settings
  • Caution Take care to minimise the light exposure on the HRI at all times


  • Set up for widefield imaging with HRI of interest
  • Use filter cube with achromatic beamsplitter with ~OD5 of absorptive ND filters
  • Focus light on glass slide (~4% reflection)
  • Record the temporal profile with 25ps gate spacings
  • Record 100 laser off background images with same integration time

Create library IRF file

  • Load measured IRF as FLIM data
  • Load camera background
  • Check for decay artefacts
  • Select small central region of image
  • Select Tools>Create IRF shift map...
  • Add to instrument calibration repository

Determining a reference lifetime

  • Load a long lifetime dye data (or a single lifetime dataset, e.g. a cell expressing GFP only)
  • Remove the background appropriately
  • IRF > Load IRF
  • Advanced tab > Select Use IRF shift map
  • Check that IRF shift map is displaying and is correct by selecting IRF shift map in the mode selector
  • In Fitting tab > set Fit reference to Fitted
  • Fit image wise both the lifetime of the sample and the reference lifetime
  • The reference lifetime obtained can then be used for fitting other dataset using the xml IRF shift map file as IRF