| ... | ... | @@ -47,17 +47,23 @@ FP = Fluorescent protein used for FCS calibration (such as monomeric GFP) |
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# Procedure
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1. Process a WT data with FA to obtain WT.res. 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 in git project of previous FCS-calibrated imaging pipeline developed by Antonio Politi. Only import, correction, and export steps are needed for the WT FA session. Then fill a WT user input in the KNIME workflow of FCSpipelineEMBL_KNIME and execute Python Source node. Use returned offset value for correction steps in further FA sessions for FP and POI.
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2. Perform three separate sessions of FA analysis: for FP, for POI, and for dye as described [here](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_fit_fcs). Do **all** intermediate FA steps (correlations calculation, correction, fitting of correlations with ACF) to obtain mFP.res, POI.res, and dye.res.
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**Important:** do not forget to save fluctuation traces with ACF fitting curves (Export all traces button) as .cof, .itr files.
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1. Process a WT data with FA to obtain WT.res. 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) you can find on git page of FCS-calibrated imaging pipeline developed by Antonio Politi. Perform import, correction, and export steps in FA. Then, fill a WT user input in the KNIME workflow of FCSpipelineEMBL_KNIME and execute Python Source node. Use returned offset value for correction steps in further FA sessions for FP and POI.
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2. Perform three separate sessions of FA analysis to obtain FP.res, POI.res, and dye.res as described [here](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_fit_fcs). Perform **all** intermediate FA steps (correlations calculation, correction, fitting of correlations with ACF) to obtain mFP.res, POI.res, and dye.res.<br><br>
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**Important notes:**
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- **Do not forget** to save fluctuation traces with ACF fitting curves (Export all traces button) as .cof, .itr files at the last step of FA analysis
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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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- 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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4. Specify parameters in the main user input. You can change any parameters or leave default values. Users have several options to calculate an effective confocal volume:
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* Using FA session for dye and then specifying the path to dye.res file in the main user input.
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* Using [FCSFitM](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/FCSFitM) provided by Antonio. In this case, users need to specify the volume in the main user input returned by FCSFitM (you can find it in focalVolume.txt). This way **requires the installation of additional software**.
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* Other strategies as described in this [article](https://www.picoquant.com/images/uploads/page/files/7351/appnote_quantfcs.pdf). After you received the value of effective confocal volume you can add it into main user input.
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4. Specify parameters in the main user input. You can change any parameters or leave default values.
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<br> Users have several options to calculate an effective confocal volume described in [technical details](https://git.embl.de/grp-almf/FCSpipelineEMBL_KNIME/-/wikis/technical-details)
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5. Fill a plot parameters input. The explanation of every parameter can be found in a KMIME description menu of the plot parameters input.
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