Abstract 202: Four dimensional quantitative label-free holographic imaging of the cell cycle in tumor cell lines
Authors: Luther Ed , Agar Jeffrey , Amiji Mansoor 
Published: August 2015

Abstract
Introduction:
Quantitative analytical systems have the ability to quantify the amount of constituents on a per entity basis. For example, flow cytometers use stoichiometric DNA stains to quantify the amount of DNA on a per cell basis, and allow discrimination of the cell cycle phases G0/G1, S-Phase, and G2/M. Quantitative imaging cytometers employ morphometric features (total DNA content, DNA maximum brightness, and nuclear area) to track the cell cycle during the mitotic phases. As cells enter mitosis, the condensation of the chromatin to form chromosomes increases the DNA density, while the nuclear area and volume decrease. Unfortunately, DNA dyes are harmful for live cells and can interfere with the results of toxicological assays. It is desirable to have a method for label-free long-term evaluation of the cell cycle in living cells.
Methods:
The Holomonitor®M4 (Phase Holographic Imaging, Lund, Sweden) is an incubator-adapted time-lapse imaging system that records interference patterns from a low power 635nm diode laser. Computer software is used to de-convolve the interference pattern at a plurality of heights to form 3-dimensional and 2-dimensional images of the sample thickness. These images are segmented and features (cellular area, optical thickness and volume, etc.) are calculated on a per cell basis. Positional information is recorded for each cell. Data can be presented as positional track charts, position vs. feature graphs, and feature vs. feature graphs. We obtained time-lapse videos using proprietary PHI software and also generated 4-dimensional plots (X-position, Y- position, optical thickness coded as the pixel intensity, and time) in ImageJ software.
Results:
In our studies of SKOV3 tumor cell lines, we found a set of cellular based features (cell volume, optical thickness, and cell area) that correlate with the DNA-based features we are accustomed to using in our traditional label-based systems. In the 4D plots, the history of each cell within the imaging area is presented for the entire evaluation time - typically 18-24 hours with 5 minute sampling increments. With a low optical thickness threshold, tracks of all of the cells are displayed. The image intensity threshold can be raised, so only mitotic events are displayed. Mitotic cells have a characteristic Y shaped tracks, appearing suddenly as the chromatin condenses, bifurcating as the daughter cells move away from each other, and then disappearing as the chromatin de-condenses. Cells blocked in mitosis present as persistent long tracks. The rapid degradation of apoptotic cells is often visible, while cells undergoing mitotic catastrophe suddenly disappear.
Conclusions:
Time-lapse label-free holographic imaging combining traditional analysis and novel display techniques is well suited for toxicological studies and visualization of drug effects on the cell cycle.
Citation Format: Ed Luther, Jeffrey Agar, Mansoor Amiji. Four dimensional quantitative label-free holographic imaging of the cell cycle in tumor cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 202. doi:10.1158/1538-7445.AM2015-202