Contrast formation (Pawley): IHC produces absorption contrast — the chromogen deposit absorbs specific wavelengths. DAB absorbs primarily in the blue region (~400-500 nm), appearing brown (transmitted red + green). Hematoxylin absorbs red and green, appearing blue-purple. The combined absorption creates the characteristic two-color appearance of H-DAB staining. Color deconvolution exploits these known absorption profiles to separate the stains computationally.
Beer-Lambert in practice: DAB deposition follows the Beer-Lambert law in optical density space: OD_DAB = ε × c × l, where c is the local chromogen concentration and l is the section thickness. This linearity makes OD a valid measure of how much chromogen was deposited — and by extension, how much target antigen is present. However, the relationship between antigen amount and DAB deposit is complicated by: (1) enzyme kinetics (HRP saturation at high antigen density), (2) development time (longer = more deposit, nonlinearly), and (3) antibody penetration (may not reach all antigen in thick sections).
Why IHC is semi-quantitative: Unlike fluorescence (where intensity has a more direct relationship to fluorophore concentration), the enzymatic amplification in IHC introduces nonlinearities. A cell with twice as much antigen doesn't necessarily produce twice the DAB deposit — the enzyme reaction has kinetic limits. This is why pathologists use ordinal scales (0/1+/2+/3+) rather than continuous measurements, and why digital pathology improves reproducibility (measuring OD consistently) but doesn't fully solve the quantitative problem.
IHC works by absorption — the brown DAB deposit blocks blue light, appearing brown in the brightfield image. Color deconvolution separates DAB from hematoxylin using their known absorption profiles. The enzyme amplification step makes IHC sensitive but also makes it non-linear — twice the target doesn't produce twice the stain, which is why scoring is semi-quantitative (0/1+/2+/3+) rather than truly quantitative.
