Pipeline example #2: Calculation of fluorescence intensity ratio plots

This is a step-by-step tutorial for creating a simple pipeline to calculate fluorescence intensity ratio plots using automatically created ROIs.

• Open an arbitrary two-channel recording
• In the Main menu use the Pipeline/New Pipeline window menu point or in the Toolbar use the button to open a new Pipeline window

In the Pipeline window:

1. Press and select I/O / Input image.
2. Press to and click on the empty workspace to add an "Input image" function.  In the Image Window parameter the default [Actual window] appears, so the input will be the top Image Window by the time of pipeline execution. Change this parameter to the caption of the Image Window to perform the pipeline on specific Image Window. This parameter can be also used to specify a Multi-Dimensiona lOpen dialog to receive input image from. Use the Description tab to toggle between the parameter list and the description of the function.
3. Press and select I/O / Get Linked Channel.
4. Press to and click on the empty workspace to add a "Get Linked Channel" function.
5. Drag the "Get Linked Channel" function by keeping the left button pressed and drop on the "Input image".
6. Repeat steps  4-5 once more.
7. Click the first "Get Linked Channel". Set the Channel (W) to 1 on the right.
8. Click the second "Get Linked Channel". Set the Channel (W) to 2 on the right.
9. Optionally, rename the "Get Linked Channel" functions to the actual channel names or numbers. For this click once the caption of the function and then keep the pointer steady over the text. Rename the function from left to right as 1 and  2.
10. Press and select Filters / Subtract Background.
11. Press to and click on an empty workspace area to add a "Subtract Background" function. In its parameter list select "Mean of pixels below percentile of max projection" as Type, because this is the most accurate method. Set 10-50 percentile as Value, depending on how much background area do the recordings have.
12. Drag the "Subtract Background" function by keeping the left button pressed and drop on the "1".
13. Right-click the "Subtract Background" function copy, and paste in an empty workspace area.
14. Drag the second "Subtract Background" function by keeping the left button pressed and drop on the "2".
15. Press and select Plotting / Automatic ROI drawing.
16. Press to and click on an empty workspace area to add a "Automatic ROI drawing" function.
17. Drag the "Automatic ROI drawing" function by keeping the left button pressed and drop on the left "Subtract Background" function.
18. Press and select Plotting / Ratio Plot.
19. Press to and click on an empty workspace area to add a "Ratio Plot" function.
20. Drag the "Ratio Plot" function by keeping the left button pressed and drop first on the "Automatic ROI drawing" function, then on the right side "Subtract Background" function. In this way channel 1 will be the numerator and channel 2 the denominator. Inputs can be reordered by dropping "Ratio Plot" function in the opposite order or by reordering inputs by clicking them in the property list.
21. Open Excel Data window using the Main Menu/Tools/Excel Data Window to collect data. Alternatively plots can be exported to text files by setting the Save name property of "Ratio Plot" to =$LoadName$.txt and Save to Yes. See automatic file naming conventions by selecting the Description tab.
22. Press to execute the pipeline on the top Image Window.  Use in the main toolbar to repeat this pipeline.
23. If using Multi-Dimensional Open dialog and  have multiple stage positions, press to execute the pipeline in all or a subset of positions.