Florence W. Patten

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.

Automated cell analysis in 2D and 3D: A comparative study

Optical projection tomographic microscopy is a technique that allows 3D analysis of individual cells. Theoretically, 3D morphometry would more accurately capture cellular features than 2D morphometry. To evaluate this thesis, classifiers based on 3D reconstructions of cell nuclei were compared with 2D images from the same nuclei. Human adenocarcinoma and normal lung epithelium cells were used. Testing demonstrated a three-fold reduction in the false negative rate for adenocarcinoma detection in 3D versus 2D at the same high specificity. We conclude that 3D imaging will potentially expand the horizon for automated cell analysis with broad applications in the biological sciences.

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

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.

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

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 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.

High resolution optical projection tomographic microscopy for 3D tissue imaging

Optical projection tomography (OPT) requires a large depth of field (DOF) of a low numerical aperture (NA) lens resulting in low resolution. However, DOF of a high NA objective can be extended by scanning the focal plane through the sample. This extended DOF image is called pseudoprojection, which is used by optical projection tomographic microscope (OPTM) for tomographic reconstruction. The advantage of OPTM is the acquisition of relatively high resolution and large depth of field concurrently. This method requires the working distance of the lens to be larger than the size of the sample, so proper lens should be chosen for samples of different sizes. In this paper, we imaged hematoxylin stained muntjac cells inside capillary tube with two different sizes. Two objective lenses with different NA are used for these two tubes. Experimental results show that resolution improves over 10 times in OPTM compared to conventional OPT, which make it possible for OPTM technique to resolve sub-cellular features for large samples. Therefore, OPTM can be used for 3D histological analysis of hematoxylin & eosin (H&E) stained biopsy specimen with sub-cellular resolution in the future.

Development of micro-optical projection tomography for 3D analysis of single cells

The Micro-Optical Projection Tomographic Microscope (μOPTM) is an instrument that is being developed for three-dimensional (3D) imaging of cells and subcellular components. The target application for the μOPTM is the early detection of lung cancer by revealing the complex 3D information about chromatin redistribution in the nucleus. The µOPTM employs a scanning objective lens (100x, N.A.=1.25) to create an extended depth-of-field image, similar to a shadowgram or projection, that we call a pseudo-projection. A large number of pseudo-projections (90+) are acquired, from which a tomographically reconstructed 3D image is computed using a filtered backprojection algorithm. The prototype μOPTM uses a single objective lens, so the object (cell) must be rotated for each new pseudo-projection. A custom microtube stage minimizes the lateral and axial motion of the sample tube during scanning and rotation so that registration between successive pseudo-projections is maintained. Image processing methods are used to correct any remaining registration errors. The media inside and outside the tube are refractive index-matched to each other and to the tube (Δnavg < 0.02). The index-matched materials are pressed between two flat parallel windows, providing a nearly distortion-free image. Custom phantoms using microspheres have been constructed and images of these 3D test targets acquired. The minimum resolvable feature size is better than 3 microns. The first 3D image of a cell using μOPTM is also shown.

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

The clinical utility of DNA ploidy as prognostic indicator is well established for cancer. Quantitative measures of DNA are possible in cell populations with flow cytometry (FCM) and individual cells with image cytometry (ICM). Because ICM can be more accurate in 3D images of absorptive stained DNA, we studied a new 3D optical microscope, the Cell-CT™. By providing quantitative cytometry of cells moving in a microcapillary tube with 3D visualization of morphology, the Cell-CT combines attributes of both FCM and ICM. Comparing Cell-CT to standard FCM, DNA ploidy measurements of Feulgen stained cultured cancer cells were made using fully automated 3D nuclear segmentation. There was a significant Spearmans rank correlation (r=0.98, p <;0.01) between the results from FCM and Cell-CT, while determining nuclear morphology of seven different cell types. We conclude that Cell-CT provides measurements of DNA content comparable to FCM and is a valuable alternative method for assessing tumorgenesis of enriched microsamples of diagnostic cells.

Quantification of relative chromatin content in flow cytometry standards using 3D OPTM imaging technique

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.