Bio-Rad EXQuest Spot Cutter - Manual (Page 145)

PDQuest User Guide
4-24
You can also use the Highlight Saturated Pixels in the Transform window to identify
regions of saturation in the image.
Faint Spots
Faint Spots on the Spots > More Edit Tools submenu and Edit Spot toolbar highlights
those spots whose signal falls below a user-defined threshold. Select the command
from the submenu or toolbar, then click in the image. The faint spots will be
highlighted in the image and the number of faint spots will be listed at the bottom of
the image window.
4.10 Spot Quantity
Spot Quantity is the total intensity of a defined spot in a gel image. This corresponds
to the amount of protein in the actual spot in the gel (see section 1.2, Digital Data and
Signal Intensity).
For spots defined using the boundary tools (section 4.6, Spot Boundary Tools), this is
simply the sum of the intensities of the pixels inside the boundary. For Gaussian
spots, it is calculated during spot detection and Gaussian fitting.
The formula for calculating the quantity of Gaussian spots in PDQuest is:
Spot height *
π
*
σ
x
*
σ
y
, where:
Spot height (also known as peak value) is the peak of the Gaussian representation of
the spot. It is measured in ODs or counts/image units
2
, depending on the type of
imaging device used.
Note: One image unit (IU) = 100 micrometers or 0.1 millimeter. This scaling factor is
used in to make the numbers in the formula more manageable.
σ
x
is the standard deviation of the Gaussian distribution of the spot in the direction of
the x axis, and
σ
y
is the standard deviation in the direction of the y axis. These are
measured in IUs.
Note that this formula results in units of OD * IU
2
for images measured in ODs, and
units of counts for images measured in counts.