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

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.

Multimodal 3D imaging of cells and tissue, bridging the gap between clinical and research microscopy.

Absorption dyes are widely used in traditional cytology and pathology clinical practice, while fluorophores and nanoparticles are more often used in biologic research. Optical projection tomographic microscopy (OPTM) is a platform technology that can image the same specimen in multiple modes in 3D, providing morphologic and molecular information concurrently and in exact co-registration. The depth-of-field of a high numerical aperture objective is extended by scanning the focal plane through the sample to generate an optical projection image. Samples of cells or tissue are brought into the OPTM instrument through a microcapillary tube filled with optical index-matching gel. Multiple optical projection images are taken from different perspectives by rotating the tube. Computed tomography (CT) algorithms are applied to these optical projection images to reconstruct 3D structure of the sample. Image segmentation and analysis based on these 3D images provide quantitative biosignatures for cancer diagnosis that represents a clear improvement over conventional 2D image analysis. In this article, we introduce the OPTM platform, optical Cell-CT, and Tissue-CT instruments, and some applications using these OPTM instruments.

Premalignant and malignant cells in sputum from lung cancer patients.

BACKGROUND: The objective of this study was to assess the frequency of premalignant and malignant cells in sputum from patients with lung cancer and to measure the dependence of these cells on cancer stage, histologic type, tumor size, and tumor location. METHODS: This analysis included 444 patients with lung cancer. First, all patients were asked to produce sputum spontaneously; then, they underwent sputum induction. Slide preparations of the sputa were screened for the presence of abnormal cells. RESULTS: Of all patients with lung cancer who had produced adequate specimens, 74.6% had sputum that was positive for premalignant or worse cells, whereas 48.7% had sputum that was positive for malignant cells alone. Surprisingly, the presence of premalignant or worse cells in sputum depended only moderately on disease stage (82.9% of stage IV cancers vs 65.9% of stage I cancers), tumor size (78.6% of tumors >2 cm vs 64.7% of tumors

Three-dimensional imaging of single isolated cell nuclei using optical projection tomography.

A method is presented for imaging single isolated cell nuclei in 3D, employing computed tomographic image reconstruction. The system uses a scanning objective lens to create an extended depth-of-field (DOF) image similar to a projection or shadowgram. A microfabricated inverted v-groove allows a microcapillary tube to be rotated with sub-micron precision, and refractive index matching within 0.02 both inside and outside the tube keeps optical distortion low. Cells or bare cell nuclei are injected into the tube and imaged in 250 angular increments from 0 to 180 degrees to collect 250 extended DOF images. After these images are further aligned, the filtered backprojection algorithm is applied to compute the 3D image. To estimate the cutoff spatial frequency in the projection image, a spatial frequency ratio function is calculated by comparing the extended depth-of-field image of a typical cell nucleus to the fixed focus image. To assess loss of resolution from fixed focus image to extended DOF image to 3D reconstructed image, the 10-90% rise distance is measured for a dyed microsphere. The resolution is found to be 0.9 microm for both extended DOF images and 3D reconstructed images. Surface and translucent volume renderings and cross-sectional slices of the 3D images are shown of a stained nucleus from fibroblast and cancer cell cultures with added color histogram mapping to highlight 3D chromatin structure.