[FLIMfit-users] FLIM-FRET image analysis

[Sean Warren]

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
Dr. Sean Warren | Research Officer
Invasion and Metastasis
Cancer Division
Garvan Institute of Medical Research
The Kinghorn Cancer Centre, 370 Victoria Street, Darlinghurst, NSW 2010

From: FLIMfit-users [mailto:flimfit-users-bounces at lists.openmicroscopy.org.uk] On Behalf Of Mayur Vadhvani
Sent: Wednesday, 22 March 2017 5:36 AM
To: flimfit-users at lists.openmicroscopy.org.uk
Cc: markosostar at gmail.com
Subject: [FLIMfit-users] FLIM-FRET image analysis

Dear All,

I am a neurobiologist/cell biologist at Institute of Biochemistry, Charite Medical University, Berlin.

I am trying to study the interaction between 2 proteins (donor GFP-tagged protein present in nucleus and cytoplasm whereas acceptor mCherry-tagged protein present only in the nucleus) using FLIM-FRET and am naive to the detailed analysis of my samples using FLIMfit.

We are using the Leica SP5 confocal system with Picoharp300 (Picoquant) at 80 MHz for acquiring our data and using FLIMfit to analyze our images.

I am going through the following steps to work on the data (sample image):
1. I assess the decay at a given pixel and import pre-defined IRF from SymphoTime64.

[Inline image 2]

2. As the pixel number is low, I use 5x5 or 7x7 smoothing (7x7 in this example).  After that I select segments in the cell (nucleus and cytoplasm respectively) and perform a mono-exponential fitting, which gives me this:
[Inline image 3]

3. Since I have FRET data, I perform a bi-exponential fit and use the Global fitting approach which gives me this:
[Inline image 4]

4. When I look at the parameters of selected segments, I get the following info:

[Inline image 6]

1- Cell 1/cytoplasm
2- Cell 1/nucleus
3- Cell 2/nucleus
4- Cell 2/cytoplasm

I had the following questions concerning my analysis:
1. I wanted to confirm whether the tau_2 values I get are the donor lifetime values for the given segments (2.35 ns in cytoplasm vs 2.22/2.20 ns in nucleus)? Because I expect FRET only in the nucleus, I would see a decrease in the lifetime values only for the nucleus as compared to the cytoplasm from the same cell (which would serve as my internal control).

2.  I don't quite understand the high tau_1 values that I observe. What could be the explanation for that?

3.Can I rely on the values presented above? I am concerned about the IRF as it is predetermined value that I am using. As suggested by Ewan McGhee in response to Marko Šoštar's query, I can try to use FITC to measure IRF of our setup.

4. Is there anything I can make my data acquisition or analysis more robust?

I would really appreciate any feedback on this.

Thank you very much.

Regards,

Mayur Vadhvani
Postdoctoral Researcher
Institute of Biochemistry
Charite Medical University
Berlin
Germany

PS:
Dear Marko,
I am also including you in the email as I am also using the Leica/PicoQuant system for my image acquisition.


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