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