| ... | ... | @@ -58,10 +58,8 @@ The block provides visualization of FCSpipelineEMBL_KNIME outputs, inspecting th |
|
|
|
- Go to save, export and report step, click FA format to save corresponding res files (**mFP.res** and **POI.res**) and save (Export all traces button) fluctuation traces with ACF fitting curves for POI and FP.
|
|
|
|
|
|
|
|
#### 3. Calculate the **effective confocal volume** by processing FCS data on the **dye**:
|
|
|
|
- Follow the same steps described in bullet point 2 to obtain **dye.res**
|
|
|
|
|
|
|
|
**or**
|
|
|
|
|
|
|
|
- Follow the same steps described in bullet point 2 to obtain **dye.res**<br>
|
|
|
|
**or**<br>
|
|
|
|
- If the effective confocal was measured without FA*, the user can specify this volume in fl in the main user input. This volume will be then used to calculate concentrations for the calibration plot. The ways to obtain the effective confocal volume outside the FCSpipelineEMBL_KNIME can be found in [technical details](https://git.embl.de/grp-almf/FCSpipelineEMBL_KNIME/-/wikis/technical-details).
|
|
|
|
|
|
|
|
|
| ... | ... | @@ -70,7 +68,7 @@ The block provides visualization of FCSpipelineEMBL_KNIME outputs, inspecting th |
|
|
|
#### Important notes regarding FA fitting step:
|
|
|
|
- There are **different fitting models** used for dye and FP&POI.
|
|
|
|
- During FA fitting step, user can use the default parameters of fit as described [here](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_fit_fcs). The parameter N can be calculated as an inverse intersection of correlations plot with the ordinate axis. This value of N can be then inserted to the table with all parameters during FA fitting step. This can help to better convergenсe of parameters used in the fitting model.
|
|
|
|
- If you calculate the effective volume with our pipeline we recommend optimizing the kappa value during the FA fitting step for dye data. For this strategy, run the fitting procedure several times with different kappa near the default meaning trying to minimize the Chi square parameter. Besides the default kappa given in the instructions of the FA fitting step, one can determine kappa experimentally.
|
|
|
|
- If you calculate the effective volume with our pipeline we recommend optimizing the kappa value during the FA fitting step for dye data. For this strategy, run the fitting procedure several times with different kappa near the default value trying to minimize the Chi-square parameter. Besides the default kappa given in the instructions of the FA fitting step, one can determine kappa experimentally.
|
|
|
|
|
|
|
|
#### 4. Prepare the following [structure of files](structure of files).
|
|
|
|
#### 5. Specify parameters in the main user input. <br>
|
| ... | ... | |
