| ... | ... | @@ -4,21 +4,21 @@ Here we explain how to install and use Fluorescence Correlation Spectroscopy (FC |
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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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#### 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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> 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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- **main user input** (where users specify general parameters)
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- **plot parameters input** (users customize the calibration plot and setting the Quality Check (QC) parameters)
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- WT user input (for calculating the background offset for correction step in 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 (calculating intensities in ROI-s, background substraction)
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- intensity calculation metanode (extracting 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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- 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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| ... | ... | @@ -28,8 +28,9 @@ Here we explain how to install and use Fluorescence Correlation Spectroscopy (FC |
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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 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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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. 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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| ... | ... | @@ -42,20 +43,27 @@ Here we explain how to install and use Fluorescence Correlation Spectroscopy (FC |
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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. Specify the channels used for collecting images. 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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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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1. Pick the point of interest in the calibration plot window to see fluctuation and correlation data from the respective FCS position.
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2. Check the statistics parameters and level of bleaching at the headings of plots.
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3. Delete points that you consider have "bad" fluctuations or quality of fitting (list points to delete in plot parameter input).
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4. The menu of the plot windows can be used to:
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* Save the image of the plot
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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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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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* Check the statistics parameters and level of bleaching at the headings of plots.
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* Move and zoom a working space
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* Adjust spacing and export values from the plot
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* Adjust the view of axes and curves
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* Adjust spacing and the view of axes and curves
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* Save the image of the plot
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8. Delete points that haven't passed Quality Check (the points with "bad" fluctuations or poor quality of fitting.
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* List the points to delete in plot parameters input. <br>
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**Important note**: The Standart Quality check does **not guarantee** to remove all "bad" fluctuations. Thus we recommend going through calibration points and remove all "bad" fluctuations manually. To delete the points in calibration plot, fill the numbers from the annotations of corresponding points into plot parameter input (points to delete). Reexecute the Python View node with calibration plot
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> This step could also help to get rid of outliers that can influence the liner parameters of the calibration line.
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#### Interactive Visualisation
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> Sensitivity of picking event can be adjusted in the plot parameters input.
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| ... | ... | @@ -64,7 +72,6 @@ Here we explain how to install and use Fluorescence Correlation Spectroscopy (FC |
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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. 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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* path to the main directory
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