UVP Life Science - Manual (Page 172)

LS Software User Guide
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Calibration Curves
• About Intensity Calibration Curves
• Optical Density
• Grey Levels
• Types of Calibration Curve
• Apply Pre-Defined Calibration Curve
• Add New Intensity Calibration Curve
• Set Amount Calibration Curve
• Change Calibration Curve Graph
About Intensity Calibration Curves
Intensity Calibration is the method of creating a mapping of input intensities to output intensities. The
input and output relationship establishes a curve. Ideally, such a mapping would be linear. However, if
finely tuned results are required, the mapping process may need to change. E.g. a pixel value of 100
might look slightly brighter, say 120. Or possibly the camera being used has a noise step of 40, which will
make all values below 40 look as dark as 0.
Two different metrics can be used in the Area Density tool to carry out analysis:
• Optical Density
• Grey Levels
The software allows users to create curves for both types of calibrations.
Optical Density
Standard Optical Density (OD) is used when the sample of interest is imaged with transmitted light. (i.e.
light going thru the sample, into the camera for imaging.) OD value of an area gives an idea of how much
light can pass through that area. If the area belongs to a sample in question, OD measures how much of
sample might be present in that area. Higher OD means less light can get through, suggesting presence
of higher quantity of sample.
Following Beer’s Law, the Optical Density of a given pixel P (say at position x,y) is calculated by LS
software in the following way:
OD = - Log [ (P(x,y) &endash; Black) / (Incident &endash; Black) ], if P(x,y) < Black
= - Log [ 1/ (Incident - Black) ], otherwise
Where
White = value of brightest white pixel in the imaging environment
Black = value of darkest black pixel in the imaging environment
Total Optical Density of an area is simply the sum total of OD values of all pixels.
Grey Levels