... | @@ -22,8 +22,7 @@ CPM = Counts per molecule<br> |
... | @@ -22,8 +22,7 @@ CPM = Counts per molecule<br> |
|
FP = Fluorescent protein used for FCS calibration (such as monomeric GFP)<br>
|
|
FP = Fluorescent protein used for FCS calibration (such as monomeric GFP)<br>
|
|
QC - Quality Check
|
|
QC - Quality Check
|
|
|
|
|
|
![wf3](uploads/3e13219973e4aabd25390e25c2c0e788/wf3.png)
|
|
![Gitlab_WF_ar](uploads/5dc4770df3256fea88df4b1467458336/Gitlab_WF_ar.jpg)
|
|
|
|
|
|
#### I - WT processing block
|
|
#### I - WT processing block
|
|
|
|
|
|
The WT processing block calculates the background offset for the correction step in the FA. See [the procedure](#procedure) below.
|
|
The WT processing block calculates the background offset for the correction step in the FA. See [the procedure](#procedure) below.
|
... | @@ -93,16 +92,24 @@ The default channel used for extracting intensities from images is Ch1. If the u |
... | @@ -93,16 +92,24 @@ The default channel used for extracting intensities from images is Ch1. If the u |
|
|
|
|
|
For the execution of all nodes at one time, press a shortcut: Shift+F7. Description of all outputs see in the [output section](#output-files)
|
|
For the execution of all nodes at one time, press a shortcut: Shift+F7. Description of all outputs see in the [output section](#output-files)
|
|
|
|
|
|
The main output node that provides interactive visualization of the calibration plot is the **Python View node**. In the Python View node users have an opportunity to:
|
|
*Execute all nodes or particular visualization nodes.<be><br>
|
|
|
|
|
|
|
|
#### 9. Visualization
|
|
|
|
1) The main output node that provides interactive visualization of the calibration plot is the **Python View node**. In the Python View node users have an opportunity to:
|
|
* 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 the next step of the procedure). The sensitivity of picking events can be adjusted in the plot parameters input
|
|
* 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 the next step of the procedure). The sensitivity of picking events can be adjusted in the plot parameters input
|
|
* Check the statistics parameters and level of bleaching in the headings of plots
|
|
* Check the statistics parameters and level of bleaching in the headings of plots
|
|
* Move and zoom a working space
|
|
* Move and zoom a working space
|
|
* Adjust spacing and the view of axes and curves
|
|
* Adjust spacing and the view of axes and curves
|
|
|
|
|
|
*Execute all nodes or particular visualization nodes.<be><br>
|
|
2) POI&FP CPM distribution node outputs the table with parameters of CPM distributions of POI and FP. See the 7th bullet point in [technical details](https://git.embl.de/grp-almf/FCSpipelineEMBL_KNIME/-/wikis/technical-details) for possible correction of calibration parameters in case of distinct modes of two distributions.
|
|
|
|
|
|
|
|
3) See the estimated concentrations of POI via FCS-calibrated imaging in the output table of **concentrations of POI** node.
|
|
|
|
|
|
|
|
4) Knime has his own Image viewer. To see raw images with concentration maps, user can right-click the Image Viewer nodes and go to View: Image Viewer. Then, double-click the image of interest.
|
|
|
|
|
|
![vis](uploads/8db7cde8c00f138270397206e1fc0218/vis.png)
|
|
![vis](uploads/8db7cde8c00f138270397206e1fc0218/vis.png)
|
|
|
|
|
|
#### 9. Quality Check <br>
|
|
#### 10. Quality Check <br>
|
|
* In the plot parameters input, list the points that haven't passed Quality Check* <br>
|
|
* In the plot parameters input, list the points that haven't passed Quality Check* <br>
|
|
**Important note**: The Standart Quality check does **not guarantee** to remove all "bad" fluctuations. Thus, we recommend going through calibration points and remove these points manually. To delete the points in the calibration plot, specify the numbers from the annotations of corresponding points in the plot parameters input (the field: points to delete).
|
|
**Important note**: The Standart Quality check does **not guarantee** to remove all "bad" fluctuations. Thus, we recommend going through calibration points and remove these points manually. To delete the points in the calibration plot, specify the numbers from the annotations of corresponding points in the plot parameters input (the field: points to delete).
|
|
* Reexecute the Python View node with a calibration plot
|
|
* Reexecute the Python View node with a calibration plot
|
... | | ... | |