Standardisation of instrumentation in plant DNA image cytometry

DNAimage cytometry is a relatively new technique for densitometric measurement of nuclear DNAcontent, which has only rarely been used in botany and thus no methodological standards exist for this method to be applied to the measurement of plant material. In the present paper we address several problems related to standardisation of DNA image cytometry, such as stability of the measuring system, linearity of optical density measurements, correction of uneven illumination of the field of view, and uniformity of integrated optical density measurement over the entire field of view. Furthermore, image processing procedures are described for mitigation of the effects of electronic noise (image averaging) and for densitometric calibration of the measuring system. We have developed a macro for plant DNAimage cytometry, using a general image analysis software package. The described quality control procedures, adopted from international medical standards for diagnostic DNAimage cytometry, were used during software development to test performance of our measurement system. Until a specific botanical consensus is reached, we recommend that the quality control standards for instrumentation described in the present paper are considered when DNA image cytometry is used for measurement of plant genome size as well as for any other image analysis-related densitometric measurement based on light microscopy