Aneuploidy is typically assessed by flow cytometry (FCM) and image cytometry (ICM). We used optical projection tomographic microscopy (OPTM) for assessing cellular DNA content using absorption and fluorescence stains. OPTM combines some of the attributes of both FCM and ICM and generates isometric high-resolution three-dimensional (3-D) images of single cells. Although the depth of field of the microscope objective was in the submicron range, it was extended by scanning the objective’s focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. These projections were later reconstructed using computed tomography methods to form a 3-D image. We also present an automated method for 3-D nuclear segmentation. Nuclei of chicken, trout, and triploid trout erythrocyte were used to calibrate OPTM. Ratios of integrated optical densities extracted from 50 images of each standard were compared to ratios of DNA indices from FCM. A comparison of mean square errors with thionin, hematoxylin, Feulgen, and SYTOX green was done. Feulgen technique was preferred as it showed highest stoichiometry, least variance, and preserved nuclear morphology in 3-D. The addition of this quantitative biomarker could further strengthen existing classifiers and improve early diagnosis of cancer using 3-D microscopy.
A potential biomarker for early diagnosis of cancer is assessment of high nuclear DNA content. Conventional
hematoxylin staining is neither stoichiometric nor reproducible. Although feulgen stain is stoichiometric, it is time
consuming and destroys nuclear morphology. We used acidic thionin stain, which can be stoichiometric and also
preserve the nuclear morphology used in conventional cytology. Fifty chicken erythrocyte nuclei singlets (CENs), diploid trout erythrocyte nuclei (TENs) and Triploid TENs were stained for 15 and 30 minutes each. After imaging with optical projection tomography microscope (OPTM), 3D reconstructions of the nuclei were processed to calculate
chromatin content. The mean of ratios of individual observations was compared with standard ratios of DNA indices
of the flow cytometry standards. Mean error, standard deviation and 97% confidence interval (CI) was computed for
the ratios of these standards. At 15 and 30 minutes, the ratio of Triploid TEN to TEN was 1.72 and 1.76, TEN to CEN
was 1.27 and 2.01 and Triploid TEN to CEN was 2.11 and 3.39 respectively. Estimates of DNA indices for all 3 types
of nuclei had less mean error at 30 minutes of staining; Triploid TEN to TEN 0.349±0.04, TEN to CEN 0.36±0.04 and Triploid TEN to CEN 0.64 ± 0.07. In conclusion, imaging of cells with thionin staining at 30 minutes and 3D reconstruction provides quantitative assessment of cell chromatin content. The addition of this quantitative feature of aneuploidy is expected to add greater accuracy to a classifier for early diagnosis of cancer based on 3D cytological imaging.