| ... | ... | @@ -19,6 +19,12 @@ CPM = Counts per molecule<br> |
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FP = Fluorescent protein used for FCS calibration (such as monomeric GFP)
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> The main nodes users work with are typed in bold.
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**Important note**: FCSpipelineEMBL_KNIME is an automated software. Only user input and visualization of data steps (see green frames) need the user's supervision.
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- **main user input** (general parameters). The explanation of every parameter can be founded on the [input parameters](https://git.embl.de/grp-almf/FCSpipelineEMBL_KNIME/-/wikis/input-parameters) page or in a KMIME description menu of the main user input.
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| ... | ... | @@ -27,25 +33,7 @@ FP = Fluorescent protein used for FCS calibration (such as monomeric GFP) |
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- WT user input (for calculating the background offset for the correction step in the Fluctuation Analyzer (FA))
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- Loading the data:
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- FP&POI, dye metanodes (collecting fluctuation data)
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- FP&POI&WT images metanode (loading images and coordinates)
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- Data processing + Quality Check:
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- Python Script (2=>1) (calculating confocal volume and concentrations)
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- intensity calculation metanode (extracting intensities in ROI-s, background subtraction)
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- joiner metanode with Quality Check (data collection for calibration plot + Quality Check)
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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 the user in plot parameters input.
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- concentration map metanode (building concentration maps from row images using calibration parameters)
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- Visualization of data
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- Python Source (POI&FP CPM distribution)
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- **Python View** (calibration plot appears 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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**Important note**: FCSpipelineEMBL_KNIME is an automated software. Only user input and visualization of data steps need the user's supervision.
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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) 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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