Advanced Topics

Creating a library IRF

Overview

  • 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

Measurement

  • 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