Quantitative characterization of cell behaviors through cell cycle progression via automated cell tracking.

Cell behaviors are reflections of intracellular tension dynamics and play important roles in many cellular processes. In this study, temporal variations in cell geometry and cell motion through cell cycle progression were quantitatively characterized via automated cell tracking for MCF-10A non-transformed breast cells, MCF-7 non-invasive breast cancer cells, and MDA-MB-231 highly metastatic breast cancer cells. A new cell segmentation method, which combines the threshold method and our modified edge based active contour method, was applied to optimize cell boundary detection for all cells in the field-of-view. An automated cell-tracking program was implemented to conduct live cell tracking over 40 hours for the three cell lines. The cell boundary and location information was measured and aligned with cell cycle progression with constructed cell lineage trees. Cell behaviors were studied in terms of cell geometry and cell motion. For cell geometry, cell area and cell axis ratio were investigated. For cell motion, instantaneous migration speed, cell motion type, as well as cell motion range were analyzed. We applied a cell-based approach that allows us to examine and compare temporal variations of cell behavior along with cell cycle progression at a single cell level. Cell body geometry along with distribution of peripheral protrusion structures appears to be associated with cell motion features. Migration speed together with motion type and motion ranges are required to distinguish the three cell-lines examined. We found that cells dividing or overlapping vertically are unique features of cell malignancy for both MCF-7 and MDA-MB-231 cells, whereas abrupt changes in cell body geometry and cell motion during mitosis are unique to highly metastatic MDA-MB-231 cells. Taken together, our live cell tracking system serves as an invaluable tool to identify cell behaviors that are unique to malignant and/or highly metastatic breast cancer cells.

Calibration of measurement sensitivities of multiple micro-cantilever dynamic modes in atomic force microscopy using a contact detection method.

An accurate experimental method is proposed for on-spot calibration of the measurement sensitivities of multiple micro-cantilever dynamic modes in atomic force microscopy. One of the key techniques devised for this method is a reliable contact detection mechanism that detects the tip-surface contact instantly. At the contact instant, the oscillation amplitude of the tip deflection, converted to that of the deflection signal in laser reading through the measurement sensitivity, exactly equals to the distance between the sample surface and the cantilever base position. Therefore, the proposed method utilizes the recorded oscillation amplitude of the deflection signal and the base position of the cantilever at the contact instant for the measurement sensitivity calibration. Experimental apparatus along with various signal processing and control modules was realized to enable automatic and rapid acquisition of multiple sets of data, with which the calibration of a single dynamic mode could be completed in less than 1 s to suppress the effect of thermal drift and measurement noise. Calibration of the measurement sensitivities of the first and second dynamic modes of three micro-cantilevers having distinct geometries was successfully demonstrated. The dependence of the measurement sensitivity on laser spot location was also experimentally investigated. Finally, an experiment was performed to validate the calibrated measurement sensitivity of the second dynamic mode of a micro-cantilever.

Control of tip-to-sample distance in atomic force microscopy: a dual-actuator tip-motion control scheme.

The control of tip-to-sample distance in atomic force microscopy (AFM) is achieved through controlling the vertical tip position of the AFM cantilever. In the vertical tip-position control, the required z motion is commanded by laser reading of the vertical tip position in real time and might contain high frequency components depending on the lateral scanning rate and topographical variations of the sample. This paper presents a dual-actuator tip-motion control scheme that enables the AFM tip to track abrupt topographical variations. In the dual-actuator scheme, an additional magnetic mode actuator is employed to achieve high bandwidth tip-motion control while the regular z scanner provides the necessary motion range. This added actuator serves to make the entire cantilever bandwidth available for tip positioning, and thus controls the tip-to-sample distance. A fast programmable electronics board was employed to realize the proposed dual-actuator control scheme, in which model cancellation algorithms were implemented to enlarge the bandwidth of the magnetic actuation and to compensate the lightly damped dynamics of the cantilever. Experiments were conducted to illustrate the capabilities of the proposed dual-actuator tip-motion control in terms of response speed and travel range. It was shown that while the bandwidth of the regular z scanner was merely a small fraction of the cantilever's bandwidth, the dual-actuator control scheme led to a tip-motion control system, the bandwidth of which was comparable to that of the cantilever, where the dynamics overdamped, and the motion range comparable to that of the z scanner.