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### Welcome to a Wiki page of FCSpipeline!
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Here we explain how to install and use Fluorescence Correlation Spectroscopy (FCS) pipeline. The FCSpipeline is an FCS-calibrated image analysis software for the interactive and automated processing of FCS data. Explanations of technical details of FCSpipeline you could find [here](technical details)
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### Welcome to a Wiki page of FCSpipelineEMBL_KNIME!
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Here we explain how to install and use FCS-calibrated image analysis pipeline. The FCSpipelineEMBL_KNIME is a software for the interactive and automated processing of FCS data.
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#### 1. [Installation](installation)
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#### 2. [Structure of workflow](#structure-of-workflow)
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#### 3. [Procedure](#procedure)
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#### 5. [Output files](#output-files)
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#### Structure of workflow
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> Explanations of technical details of FCSpipeline you could find [here](technical details)
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> The main nodes users work with are typed in bold.
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- User input:
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- **main user input** (where users specify general parameters)
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| ... | ... | @@ -21,24 +22,26 @@ Here we explain how to install and use Fluorescence Correlation Spectroscopy (FC |
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- concentration map metanode (building concentration maps from row images using calibration parameters)
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- Visualisation of data
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- Python Source (POI&FP CPM distribution)
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- **Python View** (calibration plot)
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- **Python View** (calibration plot appeares when you execute this node)
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- Table View (concentrations of POI)
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- Two Image Viewers (loaded images with info and concentration maps)
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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 can be found [here](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/home). Only import, correction, and export steps of FA session are needed. Fill a WT user input and execute Python Source node. Use returned offset value for correction steps in further FA sessions for FP and POI.
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> Remember to switch on background indicator in correction steps and then fill offset calculated in step 1
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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/home). 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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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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3. Prepare the following [structure of files](structure of files).
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4. Specify parameters in the main user input
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> You can change any parameters or leave default values. The explanation of every parameter can be founded in a description menu of the KNIME node.
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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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5. Fill a plot parameters input
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The explanation of every parameter can be founded in a KMIME description menu of the main user input.
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5. Fill a plot parameters input. The explanation of every parameter can be founded in a KMIME description menu of the plot parameters input.
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> It will implement a Quality Check by filtering the points for calibration plot according to the bounders of statistics parameters specified by user.
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| ... | ... | @@ -48,7 +51,7 @@ Here we explain how to install and use Fluorescence Correlation Spectroscopy (FC |
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7. Execute all nodes or particular visualization nodes.
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> For the execution of all nodes at one time, press a shortcut: Shift+F7.
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> For the execution of all nodes at one time, press a shortcut: Shift+F7. Execution of some nodes such as concentration maps metanode could take some time. If you don't need to build concentration maps, you can select all nodes except concentration maps metanode when executing pipeline.
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In Python View node of calibration plot users have an opportunity to:
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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 step 8 in the Procedure section)
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