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### Welcome to a Wiki page of FCS pipeline!
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Here we explain how to install and use Fluorescence Correlation Spectroscopy (FCS) pipeline. The FCSpipeline is an FCS-based calibrated image analysis software for the interactive and automated processing of FCS data.
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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.
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#### 1. [Installation](installation)
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#### 2. [Structure of workflow](#structure-of-workflow)
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| ... | ... | @@ -7,20 +7,28 @@ Here we explain how to install and use Fluorescence Correlation Spectroscopy (FC |
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#### 4. [Interactive Visualisation](#interactive-visualisation-of-calibration-plot)
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#### 5. [Output files](#output-files)
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#### Structure of workflow
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- three user inputs:
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- main user input (general parameters)
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- plot parameters input (customizing your plot and setting the QC parameters)
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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** (general parameters)
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- **plot parameters input** (customizing your plot and setting the Quality Check (QC) parameters)
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- WT user input (calculating the background offset for correction step in Fluctuation Analyzer (FA))
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- loading metanodes:
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- collecting fluctuation data (FP&POI, dye metanode)
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- loading images and coordinates (FP&POI&WT images metanode)
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- data processing nodes (Python Script (2=>1), intensity calculation metanode )
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- joiner metanode with Quality Check
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- visualization nodes
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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 (calculating intensities in ROI-s, background substraction)
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- joiner metanode with Quality Check (data collection for calibration plot + Quality Check)
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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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- 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. This step can be omitted. Process a WT data with 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.
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2. Perform two separate sessions of FA analysis: for FP and for POI. Do 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.
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1. This step can be omitted. Process a WT data with FA to obtain WT.res. Only loading and saving data steps 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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2. Perform three separate sessions of FA analysis: for FP, for POI, and for dye. Do all intermediate FA steps (correlations calculation, correction, fitting of correlations with ACF) to obtain mFP.res, POI.res, and dye.res. A detailed explanation of FA analysis procedure can be found [here](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/home).
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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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| ... | ... | @@ -32,9 +40,9 @@ Here we explain how to install and use Fluorescence Correlation Spectroscopy (FC |
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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.
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6. 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.
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6. Execute all nodes or execute different parts of pipeline sequentially
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7. Execute all nodes or execute different parts of pipeline sequentially
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> 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.
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#### Interactive Visualisation
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| ... | ... | @@ -53,9 +61,7 @@ Here we explain how to install and use Fluorescence Correlation Spectroscopy (FC |
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#### Output files
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> All outputs are saved in the main directory.
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1. **info.csv**
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info.csv is generated inside the main user input. This is the main output with all final and intermediate parameters.
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info.csv includes:
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info.csv is generated inside the main user input. This is the main output with all final and intermediate parameters. It includes:
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* diffusion coefficient of dye and confocal volume
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* the names of directories and files that are used in FCSpipeline
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