jspenc72/imagej

## imagej
# Image J Instructions
## Step 1 - Open PNG, JPG or TIF
Find and launch the OS X java application ImageJ (64 bit; menu choices and instructions were developed with the 64-bit version 1.43u of ImageJ).

ImageJicon.pngImageJ Icon

Open the gel photo. Rotate the image (if needed) so that wells are precisely horizontal across the top of the gel photo. The rotation function is accessed via the Image menu with Image→Transform→Rotate 90 degrees left/right or for smaller angle adjustments with Image→Transform→Rotate... and enter the desired angle of rotation. Positive values will rotate image in clockwise direction; negative values will rotate image in counterclockwise direction.

Use the Rectangle-selection tool (see figure below) to crop the gel image so that just lanes of interest are visible. The crop function is executed with via the Image menu with Image→Crop.

## Step 2 - Split color Channels (Optional)
Figure Legend. The ImageJ tool panel. The rectangle selection tool is in use (shaded grey on left).

Separate the gel photo into the three color channels. The split color channels function is accessed via the Image menu with Image→Color→Split Channels. Continue with the color channel with the best contrast (best signal-to-noise). The figure below shows result of Analyze→Histogram for two color channels. One histogram indicates a well balanced image; the other indicates a channel (red) that is too saturated.

## Step 3 - Analyze Gel

Figure Legend. Gel Analyzer Options panel. Navigate to this option panel with Analyze->Gels->Gel Analyzer Options.

Select lanes to create intensity profile plots. Select the outline of a lane with the rectangle selection tool. The rectangle should be placed with top edge right at the well (starting point of DNA). Make the rectangle long enough to capture all interesting bands on the bottom portion of the gel, even if such bands are not present in the first lane under consideration. Make the box a bit more narrow than the lane itself. To mark the first lane for analysis, use Analyze→Gels→Select First Lane. The shortcut for this operation to mark the first lane is command-1. Center the selection rectangle on the next lane. To mark this next lane for analysis, use Analyze→Gels→Select Next Lane. The shortcut for marking additional lanes is command-2. Repeat this step for the remaining lanes to be included in the analysis. Continue to use the shortcut command-2 even though lane indexes will be 3, 4 etc.. NOTE: If you need to start over, use the Analyze->Gels->Reset function or simply move the rectangle selection to the first lane and hit command-1. See the figure below for an example of what the gel photo will look like with lanes marked.


## Step 4 - Plot Lanes
Figure Legend. Gel photo with lanes marked. The first lane marked corresponds with the DNA ladder (marker bands). Subsequent lanes are for restriction enzyme digestion reactions.

Once the last lane has been selected, create a profile of intensity versus distance plot for each lane with Analyze→Gels→Plot Lanes (short cut: command-3). A sample intensity profile plot is shown below.
	# Image J Instructions
	## Step 1 - Open PNG, JPG or TIF
	Find and launch the OS X java application ImageJ (64 bit; menu choices and instructions were developed with the 64-bit version 1.43u of ImageJ).

	ImageJicon.pngImageJ Icon

	Open the gel photo. Rotate the image (if needed) so that wells are precisely horizontal across the top of the gel photo. The rotation function is accessed via the Image menu with Image→Transform→Rotate 90 degrees left/right or for smaller angle adjustments with Image→Transform→Rotate... and enter the desired angle of rotation. Positive values will rotate image in clockwise direction; negative values will rotate image in counterclockwise direction.

	Use the Rectangle-selection tool (see figure below) to crop the gel image so that just lanes of interest are visible. The crop function is executed with via the Image menu with Image→Crop.

	## Step 2 - Split color Channels (Optional)
	Figure Legend. The ImageJ tool panel. The rectangle selection tool is in use (shaded grey on left).

	Separate the gel photo into the three color channels. The split color channels function is accessed via the Image menu with Image→Color→Split Channels. Continue with the color channel with the best contrast (best signal-to-noise). The figure below shows result of Analyze→Histogram for two color channels. One histogram indicates a well balanced image; the other indicates a channel (red) that is too saturated.

	## Step 3 - Analyze Gel

	Figure Legend. Gel Analyzer Options panel. Navigate to this option panel with Analyze->Gels->Gel Analyzer Options.

	Select lanes to create intensity profile plots. Select the outline of a lane with the rectangle selection tool. The rectangle should be placed with top edge right at the well (starting point of DNA). Make the rectangle long enough to capture all interesting bands on the bottom portion of the gel, even if such bands are not present in the first lane under consideration. Make the box a bit more narrow than the lane itself. To mark the first lane for analysis, use Analyze→Gels→Select First Lane. The shortcut for this operation to mark the first lane is command-1. Center the selection rectangle on the next lane. To mark this next lane for analysis, use Analyze→Gels→Select Next Lane. The shortcut for marking additional lanes is command-2. Repeat this step for the remaining lanes to be included in the analysis. Continue to use the shortcut command-2 even though lane indexes will be 3, 4 etc.. NOTE: If you need to start over, use the Analyze->Gels->Reset function or simply move the rectangle selection to the first lane and hit command-1. See the figure below for an example of what the gel photo will look like with lanes marked.


	## Step 4 - Plot Lanes
	Figure Legend. Gel photo with lanes marked. The first lane marked corresponds with the DNA ladder (marker bands). Subsequent lanes are for restriction enzyme digestion reactions.

	Once the last lane has been selected, create a profile of intensity versus distance plot for each lane with Analyze→Gels→Plot Lanes (short cut: command-3). A sample intensity profile plot is shown below.