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Image Intensity Processing

38 bytes added, 10:50, 7 August 2014
Standardize keyboard shortcut formatting
If working with a stack, the ROI selected can be analyzed with the command: ''Image>Stacks>Plot Z Axis Profile''. This generates a single column of numbers - one slice intensity per row.
The top 6 rows of the column are details of the ROI. This makes sure the same ROI is not analyzed twice and allows you to save any interesting ROIs. The details are comprised of area, x-coordinate, y-coordinate, AR, roundness, and solidity of the ROI. If the ROI is a polyline>freehand ROI rather than a square>oval, it acts as if the ROI is an oval>square. The (oval) ROI can be restored by entering the details prompted by the ''Edit>Selection>Restore Selection'' (hotkey: "{{key|Ctrl }}+ {{key|Shift }}+ {{key|E"}}) command.
The results are displayed in a plot-window with the ROI details in the plot window title. The plot contains the buttons ''List, Save, Copy.'' The ''Copy'' button puts the data in the clipboard so it can be pasted into an Excel sheet. The settings for the copy button can be found under ''Edit>Options>Profile Plot Options''. Recommended settings include: ''Do not save x-values ''(prevents slice number data being pasted into Excel) and ''Autoclose'' so that you don't have to close the analyzed plot each time.
Background subtraction is needed before analysis of dual-channel ratio images. See section '''1.11 Background correction. '''The ''Ratio_Profiler'' plugin will perform ratiometric analysis of a single ROI on a dual-channel interleaved stack. The odd-slices are channel 1 images and the even slices are channel 2 images. If your two channels are opened as separate stacks, such as Zeiss, the two channels can be interleaved (mixed together by alternating between them) with the menu command "''Plugins>Stacks - Shuffling>Stack Interleaver''".
The plugin will generate a green-plot of the ratio values. Ch1÷Ch2 is the default and you can get Ch2÷Ch1 if the plugin is run with the {{key|Alt-}} key down. It will also generate a second plot of the intensities of the individual channels, Ch1 and Ch2, as well as a results table.
The first row of the results table contains values for the x, y, width and height of the ROI.
2. Open ''ROI manager'' (''Analyze>Tools>ROI manager...'') and click the "Show All" button.
3. Select the cells to be analyzed and add them to the ROI manager ("Add" button or keyboard 't' {{key|T}} key).
4. Run the plugin.
A pseudo-linescan generation of a 3-D (''x, y, t'') image. It is useful for displaying 3-D data in 2 dimensions.
A line of interest is drawn followed by the command: ''Image>Stacks>Reslice'' or with the keyboard button "{{key|/"}}. It will ask you for the line width that you wish to be averaged. It will generate a pseudo-linescan "stack" with each slice representing the pseudo-linescan of a single-pixel wide line along the line of interest. Average the pseudo-linescan "stack" by selecting ''Image>Stacks>Z-Project...'' and use the ''Average'' command. A poly-line can be utilized, but this will only generate a single pixel slice.
Fiji's default settings assume that stacks are ''z''-series rather than ''t''-series. This means that many functions related to the third-dimension of an image stack are referred to with a ''z-''. Just keep this in mind.
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