| ... | ... | @@ -39,7 +39,13 @@ Here we explain how to install and use FCS-calibrated image analysis pipeline. T |
|
|
|
2. Perform three separate sessions of FA analysis: for FP, for POI, and for dye as described [here](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/Fa_fit_fcs). Do **all** intermediate FA steps (correlations calculation, correction, fitting of correlations with ACF) to obtain mFP.res, POI.res, and dye.res.
|
|
|
|
**Important:** do not forget to save fluctuation traces with ACF fitting curves (Export all traces button) as .cof, .itr files.
|
|
|
|
3. Prepare the following [structure of files](structure of files).
|
|
|
|
4. Specify parameters in the main user input. You can change any parameters or leave default values. The explanation of every parameter can be founded in a KMIME description menu of the main user input.
|
|
|
|
4. Specify parameters in the main user input. You can change any parameters or leave default values. Users have several options to calculate an effective confocal volume:
|
|
|
|
* Using FA session for dye and then specifying the path to dye.res file in the main user input.
|
|
|
|
* Using [FCSFitM](https://git.embl.de/grp-ellenberg/FCSAnalyze/-/wikis/FCSFitM) provided by Antonio. In this case, users need to specify the volume in the main user input returned by FCSFitM (you can find it in focalVolume.txt). This way **requires the installation of additional software**.
|
|
|
|
* Other strategies as described in this [article](https://www.picoquant.com/images/uploads/page/files/7351/appnote_quantfcs.pdf). After you received the value of effective confocal volume you can add it into main user input.
|
|
|
|
|
|
|
|
|
|
|
|
The explanation of every parameter can be founded in a KMIME description menu of the main user input.
|
|
|
|
|
|
|
|
|
|
|
|
|
| ... | ... | @@ -109,4 +115,10 @@ The table with data points in calibration plot (can be used to plot the calibrat |
|
|
|
|
|
|
|
6. **map folder**
|
|
|
|
|
|
|
|
The folder contains processed concentration maps in case the concentration maps metanode was used in analysis. |
|
|
|
The folder contains processed concentration maps in case the concentration maps metanode was executed.
|
|
|
|
<br>
|
|
|
|
In order to add color representation into concentration maps obtained with pipeline user can run 2 commands in Fiji:
|
|
|
|
* run("16 colors");
|
|
|
|
* run("Calibration Bar...", "location=[Lower Right] fill=None label=White number=5 decimal=0 font=12 zoom=1 bold overlay");
|
|
|
|
|
|
|
|
 |
|
|
\ No newline at end of file |
