Examination of an accuracy of quantitative chemiluminescent imaging for histochemistry

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We examined whether the quantitation of chemiluminescent intensity is accurately measurable by image analysis instead of photon counting in tissue sections. Serial liver sections were processed for the indirect immunoperoxidase reaction with anti-CYP2B antibody or in situ hybridization with oligonucleotide probe for CYP2B2 mRNA under saturation conditions. Then, the sections were stained with the lurninol, ECL or ECL-PLUS reagent and subjected to the image analysis or photon counthing with an ARGUS-50VIM system. When a single photon hitted a pixel in CCD, pixels surrounding the hitted pixel were also stimulated. In the photon counting, such "overenphasis error" and the resulting poor spatial resolution could be eliminated by the use of a gravity detection boad. Thus, the antigen content or the relative amount of mRNA was measured succesfully by the photon counting; there was linear relationships between the content or amount and the number of counted photons. When the sections were analyzed by the image analysis, however, erroneous results due to the overenphasis error and poor resolution could not be minimized in image analysis. Furthermore, measurement should be finished before the saturation of pixels by the hitted photons. Thus, a long time measurement, which gives a high sensitivity, could not be applicable to the image analysis. A highly sensitive and an accurate quantitative chemiluminescent histochemistry is therefore difficult by the image analysis instead of photon counthing.
日本組織細胞化学会の論文

Examination of an accuracy of quantitative chemiluminescent imaging for histochemistry

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