Calculations for Slide and Small Animal Image Analysis

  • Area: Number of pixels enclosed by a shape.

  • Signal: Sum of the individual pixel intensity values (Total) for a shape minus the product of the background value and area.

    Signal = Total - (Background x Area)

  • Pixel Intensity: A value proportional to the amount of light collected from a single pixel. You can adjust the overall brightness of an image, but pixel intensities remains the same.

  • Total: Sum of the individual pixel intensities enclosed by a shape.

  • Signal-to-Noise:

    * If more than one background shape is assigned, the Background Standard Deviation is calculated by taking the square root of the pooled variance of all the pixel values from the background shapes.

Background

The background value is used to calculate background-subtracted values (like Signal) for other shapes. Signal will be calculated for shapes once a background shape is assigned.

If one background shape is assigned, the background value used for Signal calculation will be the average of all the pixel values from the background shape.

If multiple background shapes are assigned, the weighted average of all the average values from all the background shapes will be calculated and used as the background value in Signal calculations.

Weighted Average Background Calculation

n = Number of background shapes assigned
wi = Weighting factor used for the background shape. Each background shape is weighted according to the Area of pixels within the shape.
Xi = Average value of pixels within a background shape

Refine Quantification Tool

When the Refinement tool is applied to selected shape(s) on the image, pixels below a user-defined threshold intensity are eliminated from the calculation of Signal for the shape(s). This is similar to some image mask features in some software applications that hide some pixels in the image from view. However, the refinement tool affects quantification and does not change the image view.

The Standard Deviation Multiplier (Std. Dev. Multiplier) is used in this equation to calculate the Threshold Value:

When more than one background shape is assigned, the user-defined background is a weighted average of pixel intensities from all the assigned background shapes.

In general, increasing the Std. Dev. Multiplier increases the threshold and reduces the number of pixels included in the Signal value calculation for the refined quantification shape by excluding more pixels of lower intensity. Similarly, decreasing the Std. Dev. Multiplier increases the number of pixels included in the signal value calculation for the refined quantification shape.