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

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

Ethylene Induces Cell Death at Particular Phases of the Cell Cycle in the Tobacco, TBY-2 Cell Line.

It was examined whether ethylene induces program¬med cell death in a cell cycle-specific manner. Following synchronization of the tobacco TBY -2 cell line with aphidicolin and its subsequent removal, ethylene was injected into the head space of 300 cm3 culture flasks at 0 h or 3.5 h later and cells were sampled for 26 h. There were significant increases in cell mortality at G2/M in both the 0 h and 3.5 h ethylene treatments, and for the latter treatment, another peak in S-phase. The effect at G2/M was greater in the 3.5 h treatment, but was ameliorated by the simultaneous addition of silver nitrate (1.2 uM). In addition, the 3.5 h ethylene treatment resulted in a 1 h delay in the characteristic rise in the mitotic index following aphidicolin-induced synchrony. The addition of silver nitrate alone (1.2 uM), also delayed the entry of cells into mitosis but had no effect on cell cycle length compared with the controls (14 h throughout all treatments) but it induced a peak of mortality 2.5 h after its addition. Nuclear shrinkage was also a characteristic feature of dying cells at G2/M. Using Apoptag®), an in situ apoptosis detection kit, nuclear DNA fragmentation was observed in the TBY-2 cells which were often isolated on the end of a filament of normal cells. In the 3.5 h ethylene treat¬ment, a marked increase was noted in the percentage of such cells at the G2/M transition compared with the controls. Hence, the data show cell death occur¬ring at a major phase transition of the cell cycle and the observations of nuclear shrinkage, isolation of dying cells and nuclear DNA fragmentation suggest a programmed mechanism of cell death exacerbated by ethylene treatment

DNA Content Differences Between Male and Female Chicken (Gallus gallus domesticus) Nuclei and Z and W Chromosomes Resolved by Image Cytometry

Chicken red blood cells (CRBCs) are widely used as standards for DNA content determination. Cytogenetic data have shown that the Z sex chromosome is approximately twice as large as the W, so that the DNA content differs to some extent between male (ZZ) and female (ZW) chickens. Despite this fact, male and female CRBCs have been indiscriminately used in absolute genome size determination. Our work was conducted to verify whether the DNA content differences between male and female Gallus gallus domesticus “Leghorn” nuclei and ZZ/ZW chromosomes can be resolved by image cytometry (ICM). Air-dried smears stained by Feulgen reaction were used for nuclei analysis. Chicken metaphase spreads upon Feulgen staining were analyzed for obtaining quantitative information on the Z and W chromosomes. Before each capture session, we conducted quality control of the ICM instrumentation. Our results from nuclear measurements showed that the 2C value is 0.09 pg higher in males than in females. In chromosomes, we found that the Z chromosome shows 200% more DNA content than does the W chromosome. ICM demonstrated resolution power to discriminate low DNA content differences in genomes. We suggest prudence in the general use of CRBC 2C values as standards in comparative cytometric analysis. (J Histochem Cytochem 58:229–235, 2010