Michael G. Meyer

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.

Dual-modal three-dimensional imaging of single cells with isometric high resolution using an optical projection tomography microscope

The practice of clinical cytology relies on bright-field microscopy using absorption dyes like hematoxylin and eosin in the transmission mode, while the practice of research microscopy relies on fluorescence microscopy in the epi-illumination mode. The optical projection tomography microscope is an optical microscope that can generate 3-D images of single cells with isometric high resolution both in absorption and fluorescence mode. Although the depth of field of the microscope objective is in the submicron range, it can be extended by scanning the objectives focal plane. The extended depth of field image is similar to a projection in a conventional x-ray computed tomography. Cells suspended in optical gel flow through a custom-designed microcapillary. Multiple pseudoprojection images are taken by rotating the microcapillary. After these pseudoprojection images are further aligned, computed tomography methods are applied to create 3-D reconstruction. 3-D reconstructed images of single cells are shown in both absorption and fluorescence mode. Fluorescence spatial resolution is measured at 0.35 microm in both axial and lateral dimensions. Since fluorescence and absorption images are taken in two different rotations, mechanical error may cause misalignment of 3-D images. This mechanical error is estimated to be within the resolution of the system.

Automated 3-Dimensional Morphologic Analysis of Sputum Specimens for Lung Cancer Detection: Performance Characteristics Support Use in Lung Cancer Screening

The LuCED Lung Test comprises an automated 3‐dimensional morphologic analysis of epithelial cells in sputum. For each cell, 594 morphology‐based features are measured to drive algorithmic classifiers that quantitatively assess whether neoplastic cells are present. The current interim clinical study involves sputum samples from patients with known benign and malignant outcomes to assess the feasibility of LuCED as an adjunctive test after suspicious low‐dose computed tomography (LDCT) results or as an independent screening test for lung cancer.

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.

The Cell-CT 3-dimensional cell imaging technology platform enables the detection of lung cancer using the noninvasive LuCED sputum test

The war against cancer has yielded important advances in the early diagnosis and treatment of certain cancer types, but the poor detection rate and 5‐year survival rate for lung cancer has changed little over the past 40 years. Early detection through emerging lung cancer screening programs promise the most reliable means of improving mortality. Sputum cytology has been tried without success because sputum contains few malignant cells that are difficult for cytologists to detect. However, research has shown that sputum contains diagnostic malignant cells and could serve as a means of lung cancer detection if those cells could be detected and correctly characterized. Recently, the National Lung Screening Trial reported that screening using 3 consecutive low‐dose x‐ray computed tomography scans provides a 20% reduction in lung cancer mortality compared with chest x‐ray. However, this reduction in mortality comes with an unacceptable false‐positive rate that increases patient risks and the overall cost of lung cancer screening. The LuCED test for detection of early lung cancer is reviewed in the current article. LuCED is based on patient sputum that is enriched for bronchial epithelial cells. The enriched sample is then processed on the Cell‐CT, which images cells in 3 dimensions with submicron resolution. Algorithms are applied to the 3‐dimensional cell images to extract morphometric features that drive a classifier to identify cells that have abnormal characteristics. The final status of these candidate abnormal cells is established by the pathologists manual review. LuCED promotes accurate cell classification that could enable the cost‐effective detection of lung cancer. Cancer (Cancer Cytopathol) 2015;123:512–523.

Dual-mode optical projection tomography microscope using gold nanorods and hematoxylin-stained cancer cells

An optical projection tomography microscope (OPTM) can improve axial resolution by viewing a sample from different perspectives. Here, we report a dual-mode OPTM that can generate 3D images of single cancer cells in both absorption mode and polarization mode. Cancer cells were labeled with hematoxylin for absorption imaging and nanorods for polarization imaging. Absorption images can provide morphologic information, and polarization images can provide molecular information. The combination of molecular detection and 3D cytological cell analysis may help with early cancer diagnosis.

Simultaneous 3D imaging of morphology and nanoparticle distribution in single cells with the Cell-CT™ technology

The Cell-CT™ is an optical projection tomography microscope (OPTM) developed for high resolution 3D imaging of single cells based on absorption stains and brightfield microscopy. In this study we demonstrate the use of the Cell-CT™ in multi-color mode for simultaneous imaging of cellular 3D morphology and the 3D distribution of nanoparticle clusters in the cytoplasm. The ability to image cellular processes in relation to cellular compartments with a non-fluorescence 3D technology opens new perspectives for molecular research.

Nuclear cytoplasmic cell evaluation from 3D optical CT microscope images

The nuclear cytoplasmic ratio (nc-ratio) is one of the measurements made by cytologists in evaluating the state of a single cell and is defined to be the ratio of the size of the nucleus to the size of the cytoplasm. This ratio is often realized in practice by measurements on a single 2D image of a cell image acquired from a conventional microscope, and is determined by the area of the nucleus measured in the 2D image divided by the area of the cytoplasm seen to be outside of the nuclear region. It may also be defined as the ratio of the volume of the nucleus to volume of the cytoplasm, but this is not directly observable in single images from conventional 2-dimensional microscopy. We conducted a study to evaluate the variation of the 2D nc-ratio estimation due to the asymmetric architecture of cells and to compare the 2D estimates with the more precise volumetric nc-ratio estimation from 3D cell images. The measurements were made on 232 3D images of five different cell types. The results indicate that the cell orientation may cause a large amount of variation in the nc-ratio estimation and that nc-ratios computed directly from 3D images, which are independent of cell orientation, may offer a much more precise and useful measurement.

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.