Three-dimensional DNA image cytometry by optical projection tomographic microscopy for early cancer diagnosis

Abstract

Abstract. 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 highresolution 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.

Dual‐modality optical projection tomography reconstruction method from fewer views

“…Optical projection tomography (OPT), essentially an optical equivalent of X‐ray computed tomography (CT), is a noninvasive 3‐dimensional imaging approach of biological tissues or whole body of small specimen . It fills the gap of mesoscopic range of specimen sizes biometric measurement .…”

DNA ploidy measure of Feulgen-stained cancer cells using three-dimensional image cytometry

“…Cell-CT was built using a wide field optical microscope (Olympus IX71) and a custom-designed microcapillary base rotation stage [13][14][15]. Each cultured cancer cell along with TEN was stained using standard Feulgen staining technique [15,18]. The stained cells were mixed with an optical gel (Nye SmartGel OC431A-LVP) and flowed through a 50-micron inner diameter capillary tube.…”

“…The tube rotates in an oil-filled space between two flat, parallel glass slides. These parallel glass slides are later inserted into the Cell-CT for acquisition of 3D cellular images [15]. A pseudo-projection image was taken by axially scanning the objective focal plane through the sample and optically integrating all the focal plane images.…”

“…The choice of Feulgen staining method and automated 3D nuclear segmentation technique to compute the DNA ploidy of seven cultured cancer cells as an alternative to traditional two-dimensional ICM was based on a previous study [15]. Cell-CT provides 3D, submicron, isometric and high-resolution images of cellular nuclear features [15]. Since the entire volume of the nucleus is measured, there can be less uncertainty in the measurement of total DNA content.…”

“…Since the entire volume of the nucleus is measured, there can be less uncertainty in the measurement of total DNA content. In addition, Cell-CT performs multimodal imaging generating both bright field transmission image as well as a fluorescence image with exact co-registration [15]. The Cell-CT combines attributes of computed tomography (CT) imaging with slowflow cytometry.…”