We acquired multiphoton images of normal and lung adenocarcinoma cell lines in three dimensions. Image stacks of the cells were then processed to obtain nucleus-to-cytoplasm (N/C) ratios in two and three dimensions. While N/C ratios in three dimensions can be unambiguously determined from the volumetric ratios of the nucleus and cytoplasm, two-dimensional (2-D) N/C can vary depending on the axial plane selected for N/C ratio determination. We determined 2-D N/C ratios from three criteria: (1) axial position at which the nuclear area is the largest; (2) the largest 2-D N/C ratio value; and (3) axial position at the midpoint of nuclear axial position. We found that different definitions of 2-D N/C ratio will significantly affect its value. Furthermore, in general, larger variance was found in 2-D rather than three-dimensional (3-D) N/C ratios. Lack of ambiguity in definition and reduced variance suggest that 3-D N/C ratio is a better parameter for characterizing tumor cells in the clinical setting.
Histological examination has been the primary imaging modality in the diagnosis of eases such as cancer. However, since cells are three-dimensional in nature, the use of traditional nucleus to cytoplasm ratio (N/C) in two dimensions does not represent their three-dimensional structures. In this study, we used two-photon microscopy to acquired threedimensional images of normal human lung cell line Beas2B, human lung adenocarcinoma CL1-0 and CL1-5 cell lines. We determined N/C ratios in two- and three-dimensions and found that 2D N/C-ratio is more precise than 3D N/C-ratio in discriminating normal and cancer cells.