[FLIMfit-users] FLIM-FRET image analysis

[Munro, Ian]

Hi Both

If you’re both happy I feel that it would be useful if any “walkthrough” ended up accessible for others, either via e-mails on this list
or, if a video-chat, recorded and uploaded somewhere.

Thanks

Ian


On 27 Mar 2017, at 05:34, Sean Warren via FLIMfit-users <flimfit-users at lists.openmicroscopy.org.uk<mailto:flimfit-users at lists.openmicroscopy.org.uk>> wrote:

HI Mayur,

For a double exponential analysis I would advise using a measured IRF or a reference dye measurement.
A double exponential analysis is much more dependent on an accurate IRF measurement than a single exponential analysis.

Could you let me know if you are using a PMT or HyD for the FLIM measurement?

Another factor that could lead to an erroneously high long lifetime would be background light.
I would suggest making two background measurements:

1.       With the laser turned off to measure the room light/detector dark counts.
2.        A media-only measurement with no cells at the same laser power and exposure time to determine if there is background fluorescence from the media

If you can make these control measurements I’d be happy to walk you through troubleshooting your analysis.

Thanks
Sean

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