| ... | ... | @@ -42,7 +42,7 @@ The block provides visualization of FCSpipelineEMBL_KNIME outputs, inspecting th |
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# Procedure
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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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**Important:** FCSpipelineEMBL_KNIME deals with the outputs of Fluctuation Analyzer. A detailed explanation of the 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 **WT** FCS data with FA:
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| ... | ... | @@ -56,7 +56,7 @@ The block provides visualization of FCSpipelineEMBL_KNIME outputs, inspecting th |
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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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- Perform [fitting of correlations with ACF](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_fit_fcs)
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- Go to save, export and report step, 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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- Go to save, export, and report step, 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** by processing FCS data on the **dye**:
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- Follow the same steps described in bullet point 2 to obtain **dye.res**<br>
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| ... | ... | @@ -68,18 +68,20 @@ The block provides visualization of FCSpipelineEMBL_KNIME outputs, inspecting th |
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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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- 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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- During FA fitting step, users 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 value trying to minimize the Chi-square parameter. Besides the default kappa given in the instructions of the FA fitting step, one can determine kappa experimentally.
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#### 4. Prepare the following [structure of files](structure of files).
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#### 5. Specify parameters in the main user input. <br>
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Users can change the parameters or leave default ones. **Pay attention**m to the **temperature** in your experiment and the **diffusion coefficient** for your dye. User can find the table values for some of dye's diffusion coefficients [here](https://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf)
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Users can change the parameters or leave default ones. **Pay attention** to the **temperature** in your experiment and the **diffusion coefficient** for your dye. User can find the table values for some of dye's diffusion coefficients [here](https://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf)
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#### 6. Specify parameters in the plot parameters input.
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The users have the possibility to include the points with POI data into the calibration plot.
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#### 7. Specify the channels used for collecting images<br>
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The default channel used for extracting intensities from images is Ch1. If the user needs the different channel to process, go to FP&POI&WT images metanode, then open Image Reader (Table) nodes, go to Subset Selection and exclude all channels except the channel you need to process.
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| ... | ... | @@ -97,9 +99,10 @@ For the execution of all nodes at one time, press a shortcut: Shift+F7. Descript |
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* Pick the point of interest in the calibration plot window to see fluctuation and correlation data from the respective FCS position. The line can be influenced by outliers (see the next step of the procedure). The sensitivity of picking events can be adjusted in the plot parameters input
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* Check the statistics parameters and level of bleaching in the headings of plots
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* Move and zoom a working space
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* Adjust spacing and the view of axes and curves
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* Adjust spacing and the view of axes and curves<br><br>
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**Please note**: the calibration factor is calculated on FP data points. The user can include the POI data into calibration by copying the POI data files into the FP input folder.
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2) POI&FP CPM distribution node outputs the table with parameters of CPM distributions of POI and FP. See the 7th bullet point in [technical details](https://git.embl.de/grp-almf/FCSpipelineEMBL_KNIME/-/wikis/technical-details) for possible correction of calibration parameters in case of distinct modes of two distributions.
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2) POI&FP CPM distribution node outputs the table with parameters of CPM distributions of POI and FP. See the 9th bullet point in [technical details](https://git.embl.de/grp-almf/FCSpipelineEMBL_KNIME/-/wikis/technical-details) for possible correction of calibration parameters in case of distinct modes of two CPM distributions.
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3) See the estimated concentrations of POI via FCS-calibrated imaging in the output table of **concentrations of POI** node.
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| ... | ... | @@ -138,11 +141,11 @@ info.csv is generated inside the main user input. This is the main output file w |
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2. **calibration.csv**
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Here you can find the concentrations of POI in corresponding FCS positions. You can find the explanations of how concentrations are calculated on [technical details](technical details)
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Here users can find the concentrations of POI in corresponding FCS positions. You can find the explanations of how concentrations are calculated on [technical details](https://git.embl.de/grp-almf/FCSpipelineEMBL_KNIME/-/wikis/technical-details) page.
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3. **calibration_plot.png**
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The image of the final calibration plot. Here, the user has the possibility to include the POI into the plot (this option can be chosen in the "plot parameters input" node).**Please note**: the calibration factor is calculated on FP data points. The user can include the POI data into calibration by copying the POI data files into the FP input folder.
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The image of the final calibration plot.
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| ... | ... | @@ -154,11 +157,11 @@ The image of the CPM distribution. |
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5. **calibration_plot.csv**
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The table with data points in the calibration plot (can be used to plot the calibration plot with other software)
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The table with data from points in the calibration plot (can be used to plot the calibration plot with other software)
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6. **map directory**
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The folder contains processed concentration maps. In order to add color representation into concentration maps obtained with the FCSpipelineEMBL_KNIME user can run two commands in Fiji or ImageJ:
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The folder contains processed concentration maps. In order to add color representation into concentration maps obtained with the FCSpipelineEMBL_KNIME users can run two commands in Fiji&ImageJ:
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* Image -> Lookup Tables -> 16 colors
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* Analyze -> Tools -> Calibration Bar
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