Western and Gel Background

1. Lane Center Found for Analysis

   The Image Studio Lane background subtraction procedure analyzes the middle 75% of a lane to compensate for variable shape width throughout the lane. The program uses the center lane line to establish the correct area to include in analysis. The lane line is first centered approximately by the user and then optimally positioned by the software during band-finding (the software will find the exact middle of the lane, do not worry about getting the line exactly positioned).

2. Average Pixel Intensity Computed

   Next, a lane profile is created (see image below). The lane profile is the set of average pixel intensities across every row of pixels in the center 75% of the lane.

   

3. A Background Curve Is Calculated

   Background is then established by drawing a linear connection between local minima in the Intensity vs. Location graph, as long as the slope of the line is below a threshold value. Limiting the slope prevents the possibility of drawing lines through a significant portion of a signal peak. Minima that would create a slope that is too steep are skipped, and the next point that would create a line with a slope below the threshold value is used.

   Image Studio Software will not seek local minima in shapes that have been added manually. Manually added shapes are skipped, because they have been designated as important by the user.

4. Background Subtracted

   The background curve value at the top and bottom of a shape are averaged to find the background value. The background value is subtracted from the Total of the shape to find the Signal for the shape.

   

Bkgnd: The Average or Median background method computes the average or median intensity of pixels in a border around a shape and subtracts that background from the shape.

Signal: The signal for each shape is calculated by finding the Total for a shape and subtracting the product of the Bkgnd and the Area.

The area used to compute background is taken from a three pixel border around all sides of the shape by default. However, the border can be changed to the left and right or above and below the shape and the width can be adjusted from 1 - 5 pixels (see image below). The border may need to be adjusted to prevent it from overlapping a shape.

To change the border width and location:

1. Ensure the Analysis tab is visible.

2. In the Background group, click the Set Background Type list and select Average or Median.

   The Background dialog will open.

3. Change the desired settings.
4. Click OK.

Bkgnd: The User-Defined background method finds the average pixel intensity within a shape placed by the user to calculate background.

Signal: The signal is calculated by finding the Total for the shape and subtracting the product of the average intensity of the pixels in the user-defined background and the Area.

Multiple shapes can be designated as background by holding down the CTRL key and clicking each shape individually or clicking and dragging a rectangle around the shapes. If multiple shapes are selected, they will be averaged together to find the background.

Selected shapes should be designated as background by clicking Assign in the Background group of the Analysis tab. The Assign button for add-on applications may also be located in that application's Analysis tab (e.g. the Small Animal Image Analysis tab).

1. Click the small arrow at the bottom right of the Show group in the Analysis tab.

   The Image View Show Options dialog will open (see below).

   

2. In the Image View Show Options dialog, change the Display Shape Quantification Value to Total.

3. Click OK.

The displayed quantification value will be changed to None in all images.