1. Single Cell Tomography for Early Cancer Detection Vivek Nandakumar, Laimonas Kelbauskas, Roger Johnson, Deirdre Meldrum Arizona State University, Tempe, Arizona ABSTRACT Nuclear morphology is a proven biomarker for early detection of deadly diseases such as cancer. High resolution 3D cell imaging may facilitate sensitive and specific early detection. We perform optical tomographic imaging on individual, hematoxylin-stained cells to quantify variations in nuclear morphology in several cell lines spanning the neoplastic progression spectrum in esophageal cancer. Our 3D cell images are obtained by applying principles of optical projection tomography to obtain isotropic resolution of ~350 nm. Each cell image is generated with mathematical reconstruction algorithms from 500 projection images acquired over 360 ⁰. Using this technique we observe qualitative and quantitative differences in nuclear morphology between esophageal cell lines representing normal and dysplastic cells. Our results validate the superiority of 3D over 2D quantitative cytometry. RESULTS (Qualitative) CONCLUSIONS Utility of single cell tomographic imaging demonstrated for early cancer detection.. Qualitative and quantitative differences observed in nuclear morphology between cell lines. Morphological factors such as cell size, nuclear size, nucleus-cytoplasm ratio, nucleolar margination toward the nuclear membrane and total DNA content were elevated in abnormal cells. The DNA is observed to be more clumpy in abnormal cells. Variations were observed in texture of nuclear surface and chromatin. INTRODUCTION Changes in nuclear morphology are strong indicators of onset of malignancy. Computer Aided Detection (CADe) has emerged as a useful tool for early cancer diagnosis. CADe requires high resolution imagery and robust computer algorithms. SINGLE CELL TOMOGRAPHY USING Cell CT Isotropic resolution of ~350 nm. Cell imaged in suspension and not on slide. 3D image reconstructed from projection images. 500 projections acquired over 360 ⁰. Optical Projection Tomography 2 used to acquire projection image. Imaging modality: transmission (brightfield) mode, using 100x oil objective. Cells to be imaged are embedded in a thixotropic carrier gel that has same refractive index as glass. DISCUSSION Current CT capability limited to structural imaging of fixed cells. Use of Eosin to stain the cell cytoplasm is under evaluation. Incorporation of fluorescence CT modality planned to facilitate functional imaging. Significance of cell cycle in morphological changes to be assessed. More robust algorithms planned to improve accuracy of 3D nuclear morphometry. A quantitative approach to determine the most discriminatory features is currently under study. REFERENCES 1.D. Zink, A.H. Fischer, and J.A. Nickerson, Nature Reviews Cancer 4, 677 (2004). 2.M. Fauver, E.J. Seibel, J.R. Rahn et al., Optics Express 13 (11), 4210 (2005). 3.M. C. Palanca-Wessels, M. T. Barrett, P. C. Galipeau et al., Gastroenterology 114 (2), 295 (1998). MATERIALS AND METHODS Cells: Human esophageal epithelial cell lines 3. EPC (normal); CP-C (early dysplastic) & CP-D (late dysplastic) Stain: Hematoxylin, an absorption dye that heavily stains the nucleus. Sample preparation: Fix cells using CytoLyt. Stain using hematoxylin. Embed stained cells in gel. Image acquisition: Image cells using cell CT. Automated 3D cytometry: Compute nuclear morphometric features and analyze data. ACKNOWLEDGEMENTS This research is supported by NCI Center for the Convergence of Physical Science and Cancer Biology, grant number U54CA143862. Volume renderings of EPC (top), CP-C (middle) and CP-D (bottom) esophageal epithelial cells. Left images show the nuclear surface, middle images illustrate the nuclear interior, and right images depict a slab through the volume. DRAWBACKS OF CURRENT APPROACHES 0°0°45° 90°135° *Zink et al. 1 1 2 3 Normal nucleusCancerous nucleus 1 Smooth surfaceConvoluted, textured surface 2 Small nucleoliLarge nucleoli 3 Regular chromatinClumpy chromatin 4 Smooth laminaChaotic, with infoldings Normal nucleusCancerous nucleus 4 Orientation Dependence Focal Plane Dependence 2D imagery 3D imagery (confocal microscopy) Axial (along z-axis) resolution is poorer than lateral resolution (along x-,y-) by a factor of at least 2. Loss of morphological detail due to imaging on glass slide. RESULTS (Quantitative) Histograms that illustrate quantitative differences between some of the computed morphological features of the studied cell lines. One hundred 3D cell images of each cell type were used for the analysis. A total of forty features were computed using fully automated 3D image processing techniques. Significant variation is observed in the computed morphological features. Cells before and after staining with Hematoxylin. Brightfield image acquired at 50x magnification. Nuclear morphometry Image acquisition Data analysis Computer decision Rendered confocal micrograph of a mammary epithelial cell stained with syto-9 dye. Image acquired at 100x magnification. glass capillary light source objective lens #1 #500 projection image cell Morphological differences observed between studied cell lines !