Single Cell Tomography for Early Cancer Detection
Vivek Nandakumar, Laimonas Kelbauskas, Roger Johnson, Deirdre Meldrum
Arizona State University, Tempe, Arizona
SINGLE CELL TOMOGRAPHY USING Cell CT
ABSTRACT
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Nuclear morphology is a proven biomarker for early detection of deadly
• Isotropic resolution of ~350 nm.
diseases such as cancer. High resolution 3D cell imaging may facilitate
• Cell imaged in suspension and not on slide.
sensitive and specific early detection. We perform optical tomographic imaging
• 3D image reconstructed from projection images.
on individual, hematoxylin-stained cells to quantify variations in nuclear
• 500 projections acquired over 360⁰.
morphology in several cell lines spanning the neoplastic progression spectrum
• Optical Projection
in esophageal cancer. Our 3D cell images are obtained by applying principles
• Imaging modality: transmission (brightfield) mode, using 100x oil objective.
of optical projection tomography to obtain isotropic resolution of ~350 nm. Each
• Cells to be imaged are embedded in a thixotropic carrier gel that has
cell image is generated with mathematical reconstruction algorithms from 500
Tomography2
#500
RESULTS (Quantitative)
projection image
light source
Morphological differences observed between studied cell lines !
cell
used to acquire projection image.
objective lens
glass
capillary
same refractive index as glass.
projection images acquired over 360⁰. Using this technique we observe
qualitative and quantitative differences in nuclear morphology between
MATERIALS AND METHODS
esophageal cell lines representing normal and dysplastic cells. Our results
• Cells: Human esophageal epithelial cell lines3 .
validate the superiority of 3D over 2D quantitative cytometry.
• 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.
INTRODUCTION
• Changes in nuclear morphology are strong indicators of onset of malignancy.
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Normal nucleus
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Smooth surface
Convoluted, textured surface
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Small nucleoli
Large nucleoli
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Regular chromatin
Clumpy chromatin
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Smooth lamina
Chaotic, with infoldings
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Normal nucleus
Cancerous nucleus
• Stain using hematoxylin.
• Embed stained cells in gel.
• Image acquisition:
• Image cells using cell CT.
Cells before and after staining with Hematoxylin.
Brightfield image acquired at 50x magnification.
• Automated 3D cytometry:
• Compute nuclear morphometric features and analyze data.
Cancerous nucleus
*Zink et al.
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.
CONCLUSIONS
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• Computer Aided Detection (CADe) has emerged as a useful tool for early
• Utility of single cell tomographic imaging demonstrated for early cancer detection..
cancer diagnosis.
• Qualitative and quantitative differences observed in nuclear morphology between cell lines.
Nuclear
morphometry
Image
acquisition
Data
analysis
Computer
decision
RESULTS (Qualitative)
Morphological differences observed between studied cell lines !
• CADe requires high resolution imagery and robust computer algorithms.
• 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.
DRAWBACKS OF CURRENT APPROACHES
3D imagery (confocal microscopy)
2D imagery
• Orientation Dependence
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DISCUSSION
• Axial (along z-axis) resolution is
• Current CT capability limited to structural imaging of fixed cells.
poorer than lateral resolution (along
• Use of Eosin to stain the cell cytoplasm is under evaluation.
x-,y-) by a factor of at least 2.
• Incorporation of fluorescence CT modality planned to facilitate functional imaging .
• Loss of morphological detail due
• Significance of cell cycle in morphological changes to be assessed.
to imaging on glass slide.
• More robust algorithms planned to improve accuracy of 3D nuclear morphometry.
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• A quantitative approach to determine the most discriminatory features is currently under study.
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REFERENCES
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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).
• Focal Plane Dependence
Rendered confocal micrograph of a
mammary epithelial cell stained with syto-9
dye. Image acquired at 100x magnification.
ACKNOWLEDGEMENTS
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.
This research is supported by NCI Center for the Convergence of Physical Science and Cancer
Biology, grant number U54CA143862.