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**Imporatant:** FCSpipelineEMBL_KNIME deals with the outputs of Fluctuation Analyzer. A detailed explanation of FA analysis procedure including [correlations calculation](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_Load_and_Correlate) and [fitting](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_fit_fcs) can be found on the Wiki page of FCS-calibrated imaging pipeline developed by Antonio Politi.
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1. Process a WT data with FA to obtain WT.res.
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#### 1. Process a WT data with FA:
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- Perform [import, correction, and export steps in FA](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_Load_and_Correlate)
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- Perform [import, correction, and export steps in FA](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_Load_and_Correlate)
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- Click FA format to save corresponding res file as **WT.res**
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- Fill the WT user input and execute Python Source node. Use returned offset value (right-click -> Table) for correction steps in further FA sessions for FP and POI.
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2. Perform **all** intermediate steps of FA analysis for POI and FP fluctuation data to obtain mFP.res and POI.res:
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- [correlations calculation and correction](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_Load_and_Correlate)
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- [fitting of correlations with ACF](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_fit_fcs) and data export
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#### 2. Perform 2 separate FA analysis sessions for POI and FP data:
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- Perform [correlations calculation and correction](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_Load_and_Correlate)
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- Do [fitting of correlations with ACF](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_fit_fcs) and data export
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<br><be>
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- save (Export all traces button) fluctuation traces with ACF fitting curves for POI and FP.
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- Click FA format to save corresponding res files (**mFP.res** and **POI.res**) and save (Export all traces button) fluctuation traces with ACF fitting curves for POI and FP.
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#### 3. Calculate the effective confocal volume:
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**Important notes:**
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- **Do not forget** to save (Export all traces button) fluctuation traces with ACF fitting curves for POI and FP.
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#### Important notes regarding FA fitting step:
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- There are **different fitting models** used for dye and FP&POI.
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- You can use the default parameters of fit as described [here](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_fit_fcs). The parameter N can be calculated as an inverse intersection of correlations plot with the ordinate axis. Then this value of N can be inserted to the table with all parameters. This can help to better convergenсe of parameters used in the fitting model.
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- During FA fitting step, user can use the default parameters of fit as described [here](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_fit_fcs). The parameter N can be calculated as an inverse intersection of correlations plot with the ordinate axis. This value of N can be then inserted to the table with all parameters during FA fitting step. This can help to better convergenсe of parameters used in the fitting model.
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- If you calculate the effective volume with our pipeline we recommend optimizing the kappa value during the FA fitting step for dye data. For this strategy, run the fitting procedure several times with different kappa near the default meaning. Besides the default kappa given in the instructions of the FA fitting step, one can determine kappa experimentally.
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3. Prepare the following [structure of files](structure of files).
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