[FLIMfit-users] FLIM-FRET image analysis

[Mayur Vadhvani]

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.

[image: 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:
[image: 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:
[image: Inline image 4]

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

[image: 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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