<p>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.</p>
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.