[FLIMfit-users] FLIM-FRET image analysis

[Munro, Ian]

Dear Mayur 

In general I would always suggest using a measured, rather than estimated IRF.
However I’m unfamiliar with your Leica/PicoQuant system and how good the estimated IRF might be in this case.
Does anyone else know more about this system?

Regards 

Ian 


> On 21 Mar 2017, at 18:35, Mayur Vadhvani <mayurvadhvani at gmail.com> wrote:
> 
> 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.png>
> 
> 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.png>
> 
> 3. Since I have FRET data, I perform a bi-exponential fit and use the Global fitting approach which gives me this:
> <image.png>
> 
> 4. When I look at the parameters of selected segments, I get the following info:
> 
> <image.png>
> 
> 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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