http://confocal-microscopy-list.275.s1.nabble.com/Question-about-Optical-Density-tp7584144p7584147.html
If you are considering DAB staining for quantification of intensity, do consider these discussions about the DAB quantification:
There are some inherent problems while using DAB for quantification. Good luck.
> Date: Fri, 21 Aug 2015 09:59:54 -0300
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> Subject: Question about Optical Density
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> Good morning
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> I have a question about ImageJ measurement of optical density in photomicrographs obtained by digital cameras. As I understand the image captured by the camera CCD register the transmitted light. That should mean that the voltage (analogical signals) produced by the CCD is proportional to the brightness of the object. After the conversion of these signals to digital information, the brightness and gray shades of the object are translated into pixels values of gray that goes from 0 (black) to 255 (white).
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> If a pixel(a) has a gray value of 100 when compared with another pixel(b) that has a gray value of 200 we may conclude that the first one (a) is darker than the second one (b) and we may conclude that the object region corresponding to the first pixel with the gray value of 100 is also darker.
> So, when we take a measurement of the mean gray level of an area of an object (translated as Optical Density – OD), be it by using ImageJ or Axiovision (Zeiss), we should conclude that the higher values correspond to a general bright object area and the lower values correspond to a darker area of the object. In other words the value of OD has an inverse correlation with the “darkness” of the area measured.
> That reasoning may be extended to the Integrated Optical Density (IOD) that is calculated multiplying the pixel mean gray value of the area by the area of the region of the object where the measurement was taken (OD)Xarea = IOD, IOD is also the sum of the gray values of the area. That is, the lower the IOD number, the darker is the Area.
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> If we are measuring the product of an enzyme histochemical reaction or DAB deposition in a immunohistochemical DAB reaction, we may conclude that the lower the IOD, the darker is the area and consequently, the more quantity of reaction product is present the area. Of course, this relationship must be inverted if the image was originated from immunofluorescence.
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> Am I right? Is there something that I am not considering?
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> Francisco Blazquez
> School of Veterinary Medicine
> University of Sao Paulo