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.
This article presents the development of a faster control loop for oscillation amplitude regulation in tapping mode operation of atomic force microscopy. Two techniques in relation to actuation and measurement are developed, that together significantly increase the bandwidth of the control loop. Firstly, magnetic actuation is employed to directly control the tip position of the cantilever to improve both the speed and the dynamics of the positioning system. Secondly, the signal path for oscillation amplitude regulation is separated from that for topography estimation in order to eliminate measurement delay that degrades the performance of the feedback loop. As a result, the phase-crossover frequency and gain margin of the control system are both increased, leading to a faster and more stable system. Two experiments are performed, one in air and the other in aqueous solution, to compare the developed control system with a commercial one and demonstrate the improvement. The results verify that the combination of the two techniques along with other existing methods eliminates all limitations associated with the instrument for the purpose of oscillation amplitude regulation, which is therewith dictated by the bandwidth of the cantilever.
A control method, in which the tip-sample interaction force of each tapping cycle is directly regulated, is proposed for dynamic mode atomic force microscopy. It does not rely on the steady-state relationship between the cantilever’s oscillation amplitude and tip-to-sample distance, and therefore the cantilever’s transient dynamics and the time delay of rms-dc converter are irrelevant. Experimental results clearly demonstrate that the proposed method regulates the tip-sample interaction force for each tapping cycle and time delay effect is eliminated. Computer simulations also show that the proposed method reconstructs a step change in topography within two tapping cycles, independent of the cantilever’s transient dynamics.
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.
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