[FLIMfit-users] FLIM-FRET image analysis

[Mayur Vadhvani]

Dear Sean,

Thank you very much for your reply.

We are using the PMT for our FLIM measurement.

I will follow-up on the other suggestions made by you and get back to you
as soon as I have the results.

Thank you.

regards,
Mayur


On Mon, Mar 27, 2017 at 1:49 PM, Munro, Ian via FLIMfit-users <
flimfit-users at lists.openmicroscopy.org.uk> wrote:

> 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> 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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>
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