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Image Intensity Processing

47 bytes removed, 11:14, 22 March 2017
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== Brightness and Contrast ==
== Dynamic intensity vs Time analysis ==
The plugin ''Plot Z Axis Profile'' (this is the ''Z Profiler'' from Kevin (Gali) Baler (gliblr at and Wayne {{Person|Rasband }} simply renamed) will monitor the intensity of a moving ROI using a particle tracking tool. This tool can be either manual or automatic. Use the {{bc | Image | Stacks | Plot Z Axis Profile}} command.
== Getting intensity values from multiple ROIs ==
# Open confocal-series and remove the background (See Background correction)
# Generate a reference stack for the addition of ROIs. Use the ''Image {{arrow}} bc | Image | Stacks {{arrow}} | Z-project'' }} function and select the ''Average''.
# Rename this image something memorable.
# Open the ''ROI Manager'' plugin ({{bc | Analyze | Tools | Roi Manager}} or toolbar icon).
Ratiometric imaging compares the recordings of two different signals to see if there are any similarities between them. It is done by dividing one channel by another channel to produce a third ratiometric channel. This technique is useful because it corrects for dye leakage, unequal dye loading, and photo-bleaching. An example application would be measuring intracellular ion, pH, and voltage dynamics in real time.
Background subtraction is needed before analysis of dual-channel ratio images. See also the [[#Background_correction|background correction]] section. 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 {{arrow}} bc | Plugins | Stacks - Shuffling {{arrow| Stack Interleaver}} 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.
From the second row downward, the first column is the time (slice number), the second column is the Ch1 mean intensity, and the third channel is the Ch2 mean intensity and the ratio value. The stack must have its frame interval calibrated in order for the "Time" value to be in seconds. Otherwise, it is "Slices". The frame interval can be set for the stack via the menu command ''"Image {{arrowbc | Image | Properties}} Properties"''.
This table can be copied to the clipboard and pasted elsewhere with the "{{bc | Edit | Copy All}}" menu command.
To generate a reference image:
# Flatten the stack with the menu command(''Image {{arrow}} bc | Image | Stacks {{arrow}} | Z-project" }} with "Projection type: Maximum"''),
# Adjust the brightness and contrast if necessary.
# Select the new image and click the "More" button in the ROI manager. After that select "Label".
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: {{bc | 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 {{arrow}} bc | Image | Stacks {{arrow}} | 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.
# Open both the experimental image and the flat-field image.
# Click the ''Select all'' button on the flat-field image and measure the average intensity. This value, the k1 value, will appear in the results window.
# Use the ''Image Calculator plus'' plugin (Analyze {{arrow}} bc | Analyze | Tools {{arrow| Calculator plus}} Calculator plus).
# i1 = experimental image; i2 = flat-field image; k1 = mean flat-field intensity; k2 = 0. Select the "''Divide"'' operation.
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