* For Linux users: create py36.sh with the path to your Anaconda folder (**must contain bin folder**):
6. Download [fcs_pipeline_2.zip](https://git.embl.de/halavaty/fcs-pipelines-vitali-vistunou) on your computer and unzip it into the knime-workspace directory.
6. Download [the last version of FCSpipeline](https://git.embl.de/halavaty/fcs-pipelines-vitali-vistunou) on your computer and unzip it into the knime-workspace directory where all KNIME workflows are stored.
#### Structure of workflow
- two user inputs:
- main user input
- plot parameters input
- three user inputs:
- main user input (general parameters)
- plot parameters input (customizing your plot and setting the QC parameters)
- WT user input (calculating the background offset for correction step in FA)
- loading metanodes:
- collecting fluctuation data (FA WT, FA FP, FA dye metanodes)
-collecting intensities (intensity data metanode)
- data processing nodes (joiner, python scripts)
- collecting fluctuation data (FP&POI, dye metanode)
-loading images and coordinates (FP&POI&WT images metanode)
1. Prepare the following [structure of files](structure of files)
2. Specify parameters in a main user input
1. This step can be omitted. Process a WT data with Fluctuation Analyzer (FA) to obtain WT.res (only loading data and saving data steps are needed). In FCSPipeline KNIME workflow, 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.
2. Perform FA analysis including all intermediate FA steps (correlations calculation, correction, fitting of correlations with ACF) to obtain mFP.res and POI.res. **Important:** do not forget to save fluctuation traces with ACF fitting curves (Export all traces button) as **.cof, .itr files**
3. Prepare the following [structure of files](structure of files).
4. Specify parameters in a main user input
> You can change any parameters or leave default values. The explanation of every parameter can be founded in a description menu of KNIME node.
4. The default channel used for extracting intensities from images is the first one. If you want to change the channel, go to intensity data metanode, then open Image Reader (Table) node, go to Subset Selection and exclude all channels except the channel you need to process.
5. The default channel used for extracting intensities from images is the first one. If you want to change the channel, go to intensity data metanode, then open Image Reader (Table) node, go to Subset Selection and exclude all channels except the channel you need to process.
5. Execute all nodes or execute different parts of pipeline sequentially
6. Execute all nodes or execute different parts of pipeline sequentially
> For the execution of all nodes at one time, click on the green bottom (Execute all executable nodes) at the top of the KNIME window or just press a shortcut: Shift+F7.
#### Interactive Visualisation of Calibration Plot
#### Interactive Visualisation
1. Pick the point of interest in the calibration plot window to see fluctuation and correlation data from the respective FCS position.
2. Check the statistics parameters and level of bleaching at the headings of plots.
3. Delete points that you consider have "bad" fluctuations or quality of fitting (list points to delete in plot parameter input).
